Plasmodium falciparum polypeptides and methods of using same

ABSTRACT

Compositions and methods are provided for the induction of a protective immunize response in primates against a lethal challenge of  Plasmodium.

This application is a division of application Ser. No. 10/062,809, filedFeb. 1, 2002 (now abandoned), which claims the benefit of provisionalApplication Ser. No. 60/266,281, filed Feb. 1, 2001; and which also is acontinuation-in-part of application Ser. No. 09/500,376, filed Feb. 8,2000, now U.S. Pat. No. 6,855,316, the disclosures of which areexpressly incorporated by reference herein.

FIELD OF THE INVENTION

This invention is in the field of recombinant Plasmodium falciparumpolypeptides, vaccines, and vaccine formulations.

BACKGROUND OF THE INVENTION

The major merozoite surface protein of Plasmodium species has been shownto be a target of varying degrees of protective immunity against theasexual blood stages in rodent and human malaria. For example,vaccination of mice with purified P230, the major merozoite surfaceprotein of the rodent malaria Plasmodium yoelii, has resulted in reducedparasitemias in comparison to controls upon intravenous challenge with alethal dose of parasitized erythrocytes (Holder et al. 1981. Nature294:361). Mice have also been protected against P. yoelii by passivetransfer of a monoclonal antibody (Mab) specific for P230 (Majarian etal. 1984. J. Immunol. 132:3131) and against (rodent malaria) Plasmodiumchabaudi adami challenge by passive immunization with a Mab specific forthe homologous 250-kDa molecule of this plasmodium species (Lew et al.1989. Proc. Natl. Acad. Sci. USA 86:3768). The ability to conferresistance to parasite challenge by passive transfer of antibodiessuggests that antibody-mediated mechanisms play an important role inantigen-specific immunity to malaria.

Despite these findings, however, no commercially viable vaccine has beendeveloped against the major merozoite surface antigen of the major humanmalaria pathogen, Plasmodium falciparum.

For example, using naturally derived materials, such as the precursor ofthe major merozoite surface protein (MSP) alone (gp195: 195,000-200,000Da molecular species; merozoite surface protein-1 (MSP-1)), gp195 mixedwith certain of its natural processing fragments, or a naturalprocessing fragment by itself, partial protection against Plasmodiumfalciparum infection has been achieved by some researchers (Hall et al.1984. Nature 311:379; Perrin et al. 1984. J. Exp. Med. 160:441;Patarroyo et al. 1987. Vaccines 87 (Brown, Chanock, Lerner, ed.) ColdSpring Harbor Laboratory Press, CSH, NY. 117-124). An effective vaccineagainst Plasmodium falciparum should not convey merely partialprotection, i.e. a partial lowering of parasitemia, since even lowparasitemias of this organism cause serious illness. A commerciallyuseful vaccine should substantially eliminate parasitemia. It isgenerally thought that an acceptable malaria vaccine that would reduceparasitemia to a low level and, consequently, result in a substantialreduction in morbidity and mortality.

In one instance, substantially complete protection using naturalmaterials against Plasmodium falciparum challenge has been achieved inAotus monkeys, in particular by the use of a mixture of gp195 and someof its natural processing fragments obtained by affinity purificationusing a Mab, designated Mab 5.2 (Siddiqui et al. 1987. Proc. Natl. Acad.Sci. USA 84:3014). In follow up experiments using Mab 5.2 affinitypurified, parasite gp195, a correlation was found between protectionagainst infection with Plasmodium falciparum and the ability of serumantibodies to strongly inhibit parasite growth in vitro. In particular,monkeys and rabbits hyperimmunized with Mab 5.2 affinity purifiedparasite gp 195 in complete Freund's adjuvant produced antibodies thatinhibited in vitro parasite growth (Hui et al. 1987. Exp. Parasitol.64:519).

The difficulty in developing an effective naturally derived vaccine,however, has been compounded with the difficulty in developing aneffective recombinant or synthetic vaccine. Recombinant or syntheticvaccines are desirable for several reasons. They have the potential tofocus immune response(s) on the most effective portion of gp 195, animportant advantage since there may be decoy determinants in gp 195which prevent the most effective response. Also, more homogeneouspreparations are possible using recombinant techniques than inpreparations of naturally derived products. In addition, recombinant andsynthetic based vaccines avoid the potential contamination of naturallyderived gp195 with pathogens from its human source.

Although a number of investigators have designed and tested gp195-basedsynthetic peptides and recombinant products as vaccine antigens, nostrongly protective vaccine has resulted. Thus, synthetic peptidescorresponding to various segments of the N-terminal 83 kDa processingfragment of gp195 induced antibodies in rabbits which displayed only alow level of cross reactivity with asexual blood stage parasites (Cheunget al. 1986. Proc. Natl. Acad. Sci. USA 83:8328). One of these syntheticpeptides, corresponding to a non-repetitive, conserved sequence,partially protected Saimiri monkeys against Plasmodium falciparumchallenge (Cheung et al. 1986). In a vaccination study in Aotus monkeysusing an 83 kDa processing fragment-based recombinant polypeptideproduced in E. coli there was no significant difference between thecourse of infection of control animals and animals immunized with therecombinant polypeptide. In addition, very low levels of antibodiescross-reactive with native gp 195 by immunofluorescence were induced(Knapp et al. 1988. Behring Inst. Mitt. 82:349). A bacterial recombinantpolypeptide based on a fusion of two conserved regions located towardsthe amino terminus and center of the gp195 molecule induced only lowindirect fluorescent antibody (IFA) titers when used to immunize Aotusmonkeys (Herrera et al. 1990. Proc. Natl. Acad. Sci. USA 87:4017) andtwo out of five immunized animals were partially protected.

Holder et al. studied two recombinant polypeptides which corresponded toportions of the 42 kDa C-terminal processing fragment of gp195 (p42)fused to trp E and beta-galactosidase carrier sequences, respectively(Holder et al. 1988. Parasite. Immunol. 10:607). While immunized animalsproduced high antibody titers against the carrier portion of therecombinant polypeptides, much lower titers were detected against the gp195 antigen. Some of the Aotus monkeys immunized with both of theserecombinant polypeptides were partially protected against parasitechallenge.

Murphy et al. (1990. Parasitology. 2:177-183) attempted to recombinantlyproduce portions of the p42 antigen of the Wellcome isolate of gp195 ininsect host cells. gp95 is believed to exist in at least two allelicforms, of which the Wellcome isolate (“Wellcome allele”) and the MADisolate (“MAD allele”) are representative (Tanabe et al. 1987. J. Mol.Biol. 195:273). While Murphy et al. reported producing a product whichfolded in a similar manner to the natural antigen, they did not reportobtaining a purified polypeptide but only reported multiply bandedantigens speculated to have resulted from post-translational processingor degradation. No follow-up immunogenicity or efficacy studies havebeen reported using any materials obtained.

A mixture of three synthetic peptides, one peptide from the 83 kDaprocessing fragment of gp195 and two non-gp195 malaria peptides,partially to completely protected monkeys against parasite challenge; ahybrid synthetic polymer including the sequences of the three syntheticpeptides in addition to a circumsporozoite region was reported toprovide a delay or suppression of parasitemias (Patarroyo et al. 1987.Nature 328:629; Rodriguez et al. 1990. Am. J. Trop. Med. Hyg. 43:339;Patarroyo et al. 1988. Nature 332:158). Field trials of this hybrid areunder way. It is unclear whether any gp 195 epitopes in the mixture orhybrid resulted in any protection. In addition there have been tworeported studies which were unable to duplicate the prior resultsobtained using the peptide mixture (Reubush et al. 1990. Am. J. Trop.Med. Hyg. 43:355-366) or the hybrid peptide multimer (Herrera et al.1991. Abstract in the IV International Congress on Malaria andBabesiosis). Thus, there has been no gp195-based recombinant orsynthetic vaccine antigen which has been shown sufficiently effectiveagainst Plasmodium falciparum challenge.

Accordingly, it is an object of the invention to provide recombinant orsynthetic antigens, compositions comprising these antigens, and methodsof use that are effective against Plasmodium falciparum challenge.

SUMMARY OF THE INVENTION

In accordance with these objectives, the present invention provides acomposition comprising a malarial polypeptide expressed by an insectcell which contains a vector encoding the malarial polypeptide that isimmunogenic in a mammalian host.

In another aspect, the invention provides a composition comprising amalarial polypeptide and an adjuvant.

In a further aspect, the invention provides methods of inducing ananti-plasmodium immune response in a primate. The immune responsepreferably substantially reduces parasitemia in a plasmodium-infectedprimate.

In a yet another aspect, the invention provides methods of making ananti-plasmodium immunogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are schematic representations of a baculovirus p42 geneconstruct. In FIG. 1A the structure of baculovirus p42 consists of theflg5 leader sequence (flg_(L), solid box) fused to the p42 coding regionfrom amino acids Ala₁₃₃₃ to Ser₁₇₀₅. In FIG. 1B synthetic DNA (solidboxes) encodes the flg5 leader and 5′ region of p42 as aBamHI(B)/HindIII fragment. A synthetic PstI(P)/SalI(L) linker encodesthe termination codon (solid box) at the 3′ end of the construct. TheHindIII/PstI fragment was derived from cloned parasite DNA.

FIGS. 2A-B depict silver stains (lane 1) and immunoblots (lane 2) ofBVp42 (FIG. 2A) and Yp42 (FIG. 2B) electrophoresed in a 10%SDS-polyacrylamide gel. Immunoblots were reacted with rabbitanti-parasite gp195.

FIGS. 3A-B show immunoblots of purified parasite gp 195 electrophoresedunder nonreducing (FIG. 3A) or reducing (FIG. 3B) conditions and reactedwith anti-gp195 Mab 5.2 (lane 1); rabbit anti-parasite gp195 (lane 2);rabbit anti-BVp42; (#131, lane 3 and #132, lane 4); rabbit anti-Yp42(#93, lane 5 and #96, lane 6). FIG. 3C shows immunoblots of BVp42 (lane1, non-reduced; lane 2, reduced) and Yp42 (lane 3, non-reduced: lane 4,reduced) reacted with Mab 5.2.

FIGS. 4A-C are graphs depicting a competition ELISA using purified,parasite gp195 coated plates and BVp42 and gp195 inhibitors.

FIG. 5 is a graph showing ELISA titers against a BVp42 antigen ofanti-parasite gp195 sera of congenic mice.

FIG. 6 shows amino acid sequences of FUP isolate p42 (SEQ ID NO:2) andof the corresponding MAD (SEQ ID NO:3), K1 (SEQ ID NO:4) and Wellcomestrains (SEQ ID NO:5). Amino acids shown in the MAD, K1 and Wellcomestrains are those that are different from those of the FUP strain.Deletions are shown by a period. Also indicated are the sites forpost-translational modification (conserved potential N-glycosylationsites are shown as filled diamonds and non-conserved sites by opendiamonds; conserved cysteines are shown by filled circles andnon-conserved by open circles) and the beginning of the putativetransmembrane region is indicated by the arrow. Number of amino acids isaccording to Chang et al. 1988. Exp. Parasitol. 67:1.

FIGS. 7A-C show the nucleic acid sequence of the original BVp42construct (SEQ ID NO: 1) described in Example 1. The construct codes fora flg5 leader polypeptide adjacent to amino acids 1333 to 1705 of theFUP isolate gp195, as those amino acids are numbered in FIG. 6.

FIGS. 8A-D show the ELISA titer of Aotus monkeys immunized with BVp42adjuvant preparations. ELISAs were performed as described by Chang etal. 1989. The endpoint of ELISA titers for Aotus sera was determined asthe X-axis intersection of the ELISA titration curve. FIG. 8A:BVp42/MF59 (Group I); FIG. 8B: BVp42/MTP-PE+MF59 (Group II); FIG. 8C:BVp42/QS21 (Group III); FIG. 8D: BVp42/ISA51 (Group IV).

FIG. 9 shows the results of a T-cell proliferation assay of mononuclearcell cultures of Aotus vaccinated with BVp42 and either MF59 (I) orMTP-PE+MF-59 (II).

FIG. 10A shows the results of intracellular cytokine producing cells ofunstimulated cultures of Aotus vaccinated with BVp42 in adjuvants QS21(III), MTP-PE+MF59 (II), or ISA51 (IV). IFNγ: interferon-gamma; IL-4:interleukin-4; IL-10: interleukin-10.

FIG. 10B shows the results of intracellular cytokine producing cells ofBVp42 stimulated cultures of Aotus vaccinated with BVp42 in adjuvantsQS21 (III), MTP-PE+MF59 (II), or ISA51 (IV). IFNγ: interferon-gamma;IL-4: interleukin-4; IL-10: interleukin-10.

FIGS. 11A-D show the parasitemia of Aotus monkeys immunized with BVp42adjuvant preparations and administered with a lethal P. falciparumchallenge. FIG. 11A: BVp42/MF59; FIG. 11B: BVp42/QS21; FIG. 11C:BVp42/MTP-PE+MF59; FIG. 11D: BVp42/ISA51.

FIG. 12 shows the nucleotide sequence of the p42-M construct modifiedfor optimized insect cell line expression comprising nucleotides 1-1235of SEQ ID NO:6, its deduced amino acid sequence comprising amino acids1-402 of SEQ ID NO: 16, the nucleotide sequence of the p42-M (SEQ IDNO:6) construct in which the 18 nucleotides encoding the histidine tailhave been removed comprising nucleotides 1-1203 of SEQ ID NO: 17(p42-M.2) and its deduced amino acid sequence comprising amino acids1-396 of SEQ ID NO:18.

FIG. 13 shows the three-part construction of the p42-K coding region ofthe K1 allele of MSP-1 derived from the FVO strain of P. falciparum.Restriction sites used to ligate the fragments are underlined.

FIG. 14 shows the DNA sequence of BVp42-K (SEQ ID NO:7) and deducedamino acid sequence (SEQ ID NO:8).

FIG. 15 shows a silver stain of affinity chromatography purifiedBVp42-K. Lane 1: starting material; Lane 2: column filtrate; Lane 3:binding buffer flow through; Lane 4: washing buffer flow through; Lanes5-9: purified protein aliquots 3-7. Samples were electrophoresed on an11.5% polyacrylamide gel.

FIG. 16 shows a western blot of affinity chromatography purifiedBVp42-K. Lane 1: starting material; Lane 2: column filtrate; Lane 3:binding buffer flow through; Lane 4: washing buffer flow through; Lanes5-8: purified protein aliquots 3-6. Samples were electrophoresed on an11.5% polyacrylamide gel.

FIG. 17 shows a western blot of phenyl HIC and Ni-NTA chromatographypurified BVp42-K. Lane 1: pooled staring material after phenyl HICchromatography; Lane 2: column filtrate; Lane 3 is washing buffer flowthrough; Lanes 4-8: purified protein aliquots 3-7. Samples wereelectrophoresed on an 11.5% polyacrylamide gel.

FIGS. 18A and 18B show western blots of various Mabs with BVp42 (MAD20allele) and BVp42-K (K1 allele). Numbers and letters above the lanescorrespond to the specific monoclonal antibody used. Panel A: Lane 1:benchmark ladder; Lanes 2, 4, 6: BVp42-K; Lanes 3, 5, 7: BVp42. Panel B:Lane 1: benchmark ladder; Lanes 2, 4, 6, 8: BVp42-K; Lanes 3, 5, 7, 9:BVp42. Samples were electrophoresed on an 11.5% polyacrylamide gel.

FIGS. 19A and 19B show the results of N-glycosidase F digestions ofBVp42-K, BVp42, and FVO MSP-1. All samples were electrophoresed on an11.5% polyacrylamide gel.

Panel A: silver stained gel: Lanes 1-2: BVp42-K; Lanes 3-4: BVp42; Lanes5-6: FVO MSP-1. Lanes 1, 3, 5 were digested with 0.1 units ofN-glycosidase F. Lanes 2, 4, 6 are controls. Samples wereelectrophoresed on an 11.5% polyacrylamide gel. Panel B: western blot:Lanes 1-2: BVp42-K; Lanes 3-4: BVp42; Lanes 5-6: FVO MSP-1. Lanes 1, 3,5 were digested with 0.1 units of N-glycosidase F. Lanes 2, 4, 6 arecontrols.

FIG. 20 shows a western blot of recombinant baculovirus infected insectcell supernatants incubated with tunicamycin. Lanes 1-3: BVp42-K; Lanes4-6: BVp42. Lanes 1 and 4 are control wells without tunicamycin. Lanes 2and 5 contain 2.5 μg/ml tunicamycin. Lanes 3 and 6 contain 5 μg/mltunicamycin. Aliquots of cell supernatants were taken at 55 hourspost-infection and electrophoresed on an 11.5% polyacrylamide gel.

FIG. 21 depicts the general protocol for gene synthesis by PCR geneassembly. (a) overlapping 40-mer oligonucleotides encompassing theregion of interest are synthesized using standard phosphoramiditechemistry, (b) gene assembly PCR with equal concentration of eacholigonucleotide, heat-stable DNA polymerase, PCR mix (55 cycles), (c)gene amplification with 40-fold dilution of gene assembly product withheat stable DNA polymerase, 2 outside primers (23 cycles), (d)purification and restriction digestion of PCR DNA fragment product,cloning of DNA fragment into suitable plasmid vector using T4 DNAligase, and transformation into E. coli host cells (adapted from Stemmeret al., Gene 164:49-53, 1995).

FIG. 22 depicts the restriction enzyme map of reconstructed Positions ofrestriction endonuclease cleavage sites are indicated with arrowheads.Regions of nucleotide errors discovered in the nucleotide sequence ofeach construct are indicated as asterisks.

Construct I was generated from three PCR assembled fragments: Ia, Ib,and Ic. Two clones of Construct II (c1 and c2) were generated from PCRassembled fragments IIa and IIb. Construct III was produced fromrestriction fragments of c1 and c2 and a new PCR assembled fragmentConstruct IV was produced by oligonucleotide-directed site specificmutagenesis of construct III to yield the correct nucleotide sequencefor the entire p42-C6His (p42-M) gene.

FIGS. 23A and 23B depict the kinetics of protein expression of theoriginal BVp42 construct and the new BVp42-M construct. MOI=1.5 for bothconstructs. FIG. 23A shows capture ELISA quantitation of p42/p42-Mpolypeptide in crude, culture supernatants. FIG. 23B shows Coomassieblue staining of SDS-PAGE of crude, culture supernatants of p42-Mpolypeptide.

FIG. 24 shows Coomassie stain of p42-M.2 protein (hexa-histidine tagdeleted) purified from 75 ml of culture supernatant by monoclonalantibody affinity chromatography.

Lanes 1, 2, and 3 correspond to three peak column fractions. The totalpurified p42-M.2 protein yield for the three peak tubes wasapproximately 500 μg, corresponding to a protein yield of 7 mg per literof culture supernatant.

FIG. 25 shows an immunoblot of purified p42-M.2 protein (hexa-histidinetag deleted) with monoclonal antibodies specific fordisulfide-dependent, conformational determinants of native p42 (MSP1.42) (lanes 1-4) and a polyclonal antiserum against the originalrecombinant p42 encoded by BVp42. Lanes 1, 5.2; 2, G13; 3, AD9.1; 4,G14(S.L.) and 5, polyclonal rabbit anti-MSP 1.42.

FIG. 26 shows Coomassie blue staining of SDS-PAGE of p42-M.2(hexa-histidine tag deleted) polypeptide material at various stagesduring chromatography. Column 1: Phenyl Sepharose. Column 2: CibachronBlue Sepharose.

FIG. 27 shows the codon preferences for the nucleotide sequence of thep42-M and p42-M.2 (hexa-histidine tag deleted) constructs modified foroptimized insect cell line Trichoplusia ni (HIGH FIVE™) expression.

DETAILED DESCRIPTION OF THE INVENTION

BVp42 antigen of Plasmodium falciparum gp195 induces antibodies thatstrongly, if not substantially completely, inhibit parasite growth andforms the basis of a vaccine. The antibodies are extensivelycrossreactive with different parasite strains, and have been found tostrongly or completely inhibit parasite growth of heterologous parasitesto the same degree as homologous parasites. BVp42, as described herein,is a variant of the natural p42 processing fragment of gp195 that hasbeen recombinantly expressed in insect cells using a baculovirusexpression vector. The term “BVp42” as used herein to refer to the p42amino acid sequence as characteristically produced in insect cells, inparticular in sf9 insect cells or HIGH FIVE™/BTI-TN-5B1-4 cells. Theamino acid sequence of p42 of different isolates is shown in FIG. 6,i.e. amino acids nos. 1333 to 1726 in the FUP isolate, 1308 to 1701 inthe MAD isolate, 1264 to 1640 in the Wellcome isolate, and 1255 to 1631in the K1 isolate (the numbers refer to the amino acids of the precursormolecule, gp195). The term “p42” as used herein refers to thecorresponding sequences in other isolates as well. A preferredembodiment of the invention includes only the amino acids Ala₁₃₃₃ toSer₁₇₀₅ of the FUP isolate (Chang et al. Exp. Parasitol. 67:1), or thecorresponding amino acids of other isolates (e.g. Ala₁₃₀₈ to Ser₁₆₈₀ ofthe MAD isolate (Mackay et al. 1985. EMBO J. 4:3823-3829), Ala₁₂₆₄ toSer₁₆₁₉ of the Wellcome isolate (Holder et al. 1985. Nature317:270-273), Ala₁₂₅₅ to Ser₁₆₁₀ of the K1 isolate (Mackay et al. 1984.EMBO J. 4:3823-3829). (The numbering of these amino acids alsocorresponds to that shown in FIG. 6.) The antigen of this embodimentpreferably deletes the anchor sequence at the C-terminus of p42,allowing easier recovery of the product because it is secreted from thehost cells.

The amino acid sequences of isolates bearing the same designation mayvary somewhat, as may the DNA sequences coding for those isolates.Similarly, the numberings of the amino acid and DNA sequences in otherpublications may differ from the numberings shown herein. These andother aspects of the invention are more fully described below. Forexample, a particular amino acid and DNA sequence of a FUP isolatecorresponding to amino acids Ala₁₃₃₃ to Ser₁₇₀₅ (SEQ ID NO:2) (as shownin FIG. 6) is described in the examples below and has the sequencesshown in FIG. 7 (SEQ ID NO: 1).

Accordingly, by “p42 polypeptide” herein is meant a polypeptidecomprising a p42 amino acid sequence, including fragments and variantsthereof, of the Plasmodium major merozoite surface protein (gp195). Thep42-M polypeptide is included within the definition of a p42 polypeptideand comprises a p42 polypeptide encoded by a p42-M nucleic acid sequence(see e.g., SEQ ID NO: 6) which differs from the p42 nucleic acids (seee.g., SEQ ID NO: 1). The “p42-M nucleic acid” comprises a nucleic acidin which one or more codons have been substituted with codons encodingthe same or similar amino acid (see e.g., the amino substitutions asdescribed herein) wherein the p42-M mRNA transcript is preferentiallyrecognized by the tRNAs present in the insect host cell expressionsystem (see, e.g., FIG. 27 which discloses the codons preferentiallyused by insect host cell Trichoplusia ni). One p42-M amino acidsequence, as set forth in SEQ ID NO: 16, differs from the amino acidsequence encoded by the p42 nucleic acid sequence set forth in FIG. 7,by a single residue at position 257. The p42-M.2 polypeptide is furtherincluded within the definition of a p42 polypeptide and comprises a p42polypeptide encoded by a p42-M.2 nucleic acid (see e.g., SEQ ID NO: 17)wherein the histidine tail of p42 or p42-M has been deleted (see e.g.,amino acid residues 1-392 of SEQ ID NO: 18).

By “polypeptide” and grammatical equivalents herein are meant proteins,oligopeptides, and peptides, derivatives and analogs, including proteinscontaining non-naturally occurring amino acids and amino acid analogs,and peptidomimetic structures. The side chains may be in either the (R)or the (S) configuration. In a preferred embodiment, the amino acids arein the (S) or L-configuration. In some embodiments, for example when thep42 polypeptides are made synthetically, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

In a preferred embodiment, the p42 polypeptide is isolated. By “isolatedpolypeptide” herein is meant a polypeptide which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least about 15 residuesof N-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated p42 polypeptide includes the p42 polypeptide expressedby recombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedp42 polypeptide will be prepared by at least one purification step.

In a preferred embodiment, a p42 polypeptide comprises a native sequencep42-polypeptide. A “native sequence” p42 polypeptide comprises apolypeptide having the same amino acid sequence as a p42 polypeptidederived from nature. The term “native sequence” specifically encompassesnaturally-occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants andnaturally-occurring Plasmodium species variants. In one embodiment ofthe invention, the native sequence p42 is a mature or full-length nativesequence p42 polypeptide.

The p42 “extracellular domain” refers to a form of the p42 polypeptidewhich is essentially free of the transmembrane (Haldar et al. 1985. J.Biol. Chem. 260(8):4969-4974) and cytoplasmic domains. Ordinarily, a p42polypeptide extracellular domain will have less than about 1% of suchtransmembrane and/or cytoplasmic domains and preferably, will have lessthan about 0.5% of such domains. It will be understood that anytransmembrane domain(s) identified for the p42 polypeptides of thepresent invention are identified pursuant to criteria routinely employedin the art to identify that type of hydrophobic domain. In a preferredembodiment the transmembrane domain is identified as that portion of thep42 polypeptide that anchors the p42 polypeptide to a membrane. In analternative embodiment, in the absence of the transmembrane domain, thep42 polypeptide is not anchored to a membrane and is therefore notassociated with the cell in which the p42 polypeptide is expressed.Accordingly, in a preferred embodiment the p42 polypeptide extracellulardomain comprises amino acids from about Ala₁₃₃₃ to about Ser₁₇₀₅ of theFUP isolate (SEQ ID NO:2) (Chang et al. Exp. Parasitol. 67:1), or thecorresponding amino acids of other isolates (e.g. Ala₁₃₀₈ to Ser₁₆₈₀ ofthe MAD isolate (SEQ ID NO:4) (Mackay et al. 1985. EMBO J. 4:3823-3829),Ala₁₂₆₄ to Ser₁₆₁₉ of the Wellcome isolate (SEQ ID NO:5) (Holder et al.1985. Nature 317:270-273), Ala₁₂₅₅ to Ser₁₆₁₀ of the K1 isolate (SEQ IDNO:3) (Mackay et al. 1984. EMBO J. 4:3823-3829).

In one embodiment, p42 polypeptides are identified by having substantialamino acid sequence homology with the amino acid sequences providedherein. In another embodiment, p42 polypeptide is identified as beingencoded by a nucleic acid having substantial nucleic acid sequencehomology with the nucleic acid sequences that are provided herein orwith the nucleic acid sequences that encode the amino acid sequencesprovided herein. By “homology” herein is meant sequence similarity andidentity with identity being preferred. Such sequence identity orsimilarity can be based upon the overall amino acid or nucleic acidsequence.

In a preferred embodiment, a polypeptide is a p42 polypeptide as definedherein if the overall sequence identity of the amino acid sequences ofFIG. 6 is preferably greater than about 60%, more preferably greaterthan about 70%, even more preferably greater than about 80% and mostpreferably greater than 90%. In some embodiments the sequence identitywill be as high as about 93 to 95 or 98%.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman. 1981. Adv. Appl. Math. 2:482, which isexpressly incorporated by reference, by the sequence identity alignmentalgorithm of Needleman & Wunsch. 1970. J. Mol. Biol. 48:443, which isexpressly incorporated by reference, by the search for similarity methodof Pearson & Lipman. 1988. PNAS USA 85:2444, which is expresslyincorporated by reference, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al. 1984.Nucl. Acid Res. 12:387-395, all of which are expressly incorporated byreference, preferably using the default settings, or by inspection.Preferably, percent identity is calculated by FastDB based upon thefollowing parameters: mismatch penalty of 1; gap penalty of 1; gap sizepenalty of 0.33; and joining penalty of 30, “Current Methods in SequenceComparison and Analysis,” Macromolecule Sequencing and Synthesis,Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss,Inc., which is expressly incorporated by reference.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle. 1987. J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins &Sharp. 1989. CABIOS 5:151-153, all of which are expressly incorporatedby reference. Useful PILEUP parameters include a default gap weight of3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993), all of which are expresslyincorporated by reference. A particularly useful BLAST program is theWU-BLAST-2 program which was obtained form Altschul et al., Method inEnzymology, 266: 460-480 (1996), all of which are expressly incorporatedby reference. WU-BLAST-2 uses several search parameters, most of whichare set to the default values. The adjustable parameters are set withthe following values: overlap span=1, overlap fraction=0.125, wordthreshold (T)=11. The HSP S and HSP S2 prorameters are dynamic valuesand are established by the program itself depending upon the compositionof the particular sequence and composition of the particular databaseagainst which the sequence of interest is being searched; however, thevalues may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402, which is expressly incorporatedby reference. Gapped BLAST uses BLOSUM-62 substitution scores; thresholdT parameter set to 9; the two-hit method to trigger ungapped extensions;charges gap lengths of k a cost of 10+k; X_(u) set to 16, and X_(g) setto 40 for database search stage and to 67 for the output stage of thealgorithms. Gapped alignments are triggered by a score corresponding to˜22 bits.

A % amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the coding sequence of the polypeptides identified herein isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleotide residues in the coding sequenceof p42 polypeptide. A preferred method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids or nucleic acid residues than the sequences in thefigures, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acid or nucleic acid residues in relation to the total number ofresidues. Thus, for example, sequence identity of sequences shorter thanthat shown in FIG. 1, as discussed below, will be determined using thenumber of amino acids in the shorter sequence, in one embodiment. Inpercent identity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as, insertions, deletions,substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedherein for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “longer”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

As will be appreciated by those skilled in the art, the sequences of thepresent invention may contain sequencing errors. That is, there may beincorrect nucleosides, frameshifts, unknown nucleosides, or other typesof sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

The p42 polypeptides of the present invention may be shorter or longerthan the amino acid sequences shown in the figures. Thus, in a preferredembodiment, included within the definition of p42 polypeptides areportions or fragments of the amino acid sequences provided herein. Inone embodiment, fragments of p42 polypeptides are considered p42polypeptides if a) they share at least one antigenic epitope; b) have atleast the indicated sequence identity; and/or c) preferably have p42polypeptide immunologic activity as defined herein. The nucleic acidsencoding the p42 polypeptides also can be shorter or longer than thesequences in the figures.

In addition, as is more fully outlined below, p42 polypeptides can bemade that are longer than those depicted in the figures. For example, bythe addition of an epitope or purification tags, the addition of otherfusion sequences, or the elucidation of additional coding and non-codingsequences. As described below, the fusion of a p42 polypeptides to apolypeptide, such as a flg5 leader polypeptide, is particularlypreferred.

p42 polypeptides may also be identified as encoded by p42 nucleic acidswhich hybridize to the sequences depicted in the figures or to nucleicacid sequences that encode the amino acid sequences depicted in thefigures, or the complement thereof, as outlined herein. Hybridizationconditions are further described below.

In a preferred embodiment, p42 polypeptide must share at least oneepitope or determinant with the full length protein. By “epitope” or“determinant” herein is meant a portion of a protein which will generateand/or bind an antibody. Thus, in most instances, antibodies made tosmaller p42 polypeptides will be able to bind to the full lengthprotein. In another embodiment, antibodies made to native p42polypeptide will bind to smaller p42 polypeptides, provided that thesmaller polypeptides contain an epitope found on the full-lengthpolypeptide that is recognized by the antibodies made to native p42polypeptide. Accordingly, in a preferred embodiment p42 polypeptide isimmunogenic. By “immunogenic” or “immunogen” and grammatical equivalentsherein is meant a substances that induces or evokes an immune response,such as a cell-mediated and/or humoral (antibody) immune response, in amammal. In a preferred embodiment the immune response substantiallyreduces the symptoms and manifestations associated with plasmodiuminfection, as described herein.

In the case of the nucleic acid, the overall sequence identity of thenucleic acid sequence is commensurate with amino acid sequence identitybut takes into account the degeneracy in the genetic code and codon biasof different organisms. Accordingly, the nucleic acid sequence identitymay be either lower or higher than that of the protein sequence. Thusthe sequence identity of the nucleic acid sequence as compared to thenucleic acid sequence of the figures is preferably greater than 75%,more preferably greater than about 80%, particularly greater than about85% and most preferably greater than 90%. In some embodiments thesequence identity will be as high as about 93 to 95 or 98%.

In a preferred embodiment, a p42 nucleic acid encodes a p42 polypeptide.As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the p42 polypeptides of the present invention. Thus,having identified a particular amino acid sequence, those skilled in theart could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the p42 polypeptide.

In one embodiment, a p42 polypeptide is identified as being encoded by anucleic acid that hybridizes under high stringency to the nucleic acidssequence shown in the figures, or their complement. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et at., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences specifically hybridize at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology-Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993), which is expresslyincorporated by reference. Generally, stringent conditions are selectedto be about 5-10° C. lower than the thermal melting point (T_(m)) forthe specific sequence at a defined ionic strength, pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at about pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.about 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

As used herein and further defined below, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA, andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in the figures also include the complement of thesequence.

By the term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro or in a cell in culture, in general, by themanipulation of nucleic acid by endonucleases and/or exonucleases and/orpolymerases and/or ligases and/or recombinases, to produce a nucleicacid not normally found in nature. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention.

The p42-M nucleic acids (see e.g., nucleotides 1-1232 of SEQ ID NO:6)are included within the definition of p42 nucleic acids and comprisenucleic acids encoding p42 and p42-M polypeptides (see e.g., amino acidresidues 1-402 of SEQ ID NO: 16) which differ from p42 nucleic acids(see e.g., SEQ ID NO: 1) by the substitution of one or more codons withcodons encoding the same or similar amino acid (see e.g., the aminosubstitutions as described herein) wherein the p42-M mRNA transcript ispreferentially recognized by the tRNAs present in the insect host cellexpression system (see, e.g., FIG. 27 which discloses the codonspreferentially used by insect host cell Trichoplusia ni). This resultsin enhanced translation of the p42-M mRNA transcripts and enhancedproduction of the p42-M polypeptide. The p42-M.2 nucleic acids (seee.g., nucleotides 1-1200 of SEQ ID NO: 17) are further included withinthe definition of p42 nucleic acids and comprise nucleic acids encodingp42 and p42-M.2 polypeptides (see e.g., amino acid residues 1-392 of SEQID NO:18) wherein the 18 nucleotides encoding the histidine tail havebeen deleted.

The p42-M and p42-M.2 nucleic acid sequences exhibit one or more of theproperties of optimizing recombinant polypeptide expression in insecthost cells, of expressing higher recombinant polypeptide concentrationsof immunogen in culture supernatants, and of expressing a morehomogenous (or purified) recovered p42 immunogen. In a preferredembodiment, the p42-M nucleic acids exhibit a 1-2 fold increase in p42-Mpolypeptide expression levels relative to p42 nucleotide expression ofp42 polypeptides, more preferably the p42-M nucleic acids exhibit a 2-3fold increase in p42-M polypeptide expression levels relative to p42nucleotide expression of p42 polypeptides, even more preferably thep42-M nucleic acids exhibit a 3-4 fold increase in p42-M polypeptideexpression levels relative to p42 nucleotide expression of p42polypeptides, and most preferably the p42-M nucleic acids exhibit atleast a 4 fold increase in p42-M polypeptide expression levels relativeto p42 nucleotide expression of p42 polypeptides.

In another preferred embodiment, the p42-M.2 nucleic acids exhibit a 1-3fold increase in p42-M.2 polypeptide expression levels relative to p42nucleotide expression of p42 polypeptides, more preferably the p42-M.2nucleic acids exhibit a 4-5 fold increase in p42-M.2 polypeptideexpression levels relative to p42 nucleotide expression of p42polypeptides, even more preferably the p42-M.2 nucleic acids exhibit a6-7 fold increase in p42-M.2 polypeptide expression levels relative top42 nucleotide expression of p42 polypeptides, and most preferably thep42-M.2 nucleic acids exhibit at least an 8 fold increase in p42-M.2polypeptide expression levels relative to p42 nucleotide expression ofp42 polypeptides.

In still another preferred embodiment, the p42-M and p42-M.2 nucleicacids are expressed in a recombinant baculovirus (BV) possessing anoptimized promoter and translation initiation region operably linked tothe p42-M or p42-M.2 nucleic acid. In a preferred embodiment, anoptimized promoter and translation initiation region are operably linkedto a p42-M.2 nucleic acid.

In still another preferred embodiment, the p42-M and p42-M.2 nucleicacids are expressed in a recombinant baculovirus (BV) possessing anoptimized promoter and translation initiation region operably linked tothe p42-M and p42-M.2 nucleic acid. In still another embodiment, Tn5(Trichoplusia ni or HIGH FIVE™) host cells are transformed withbaculovirus expressing the p42-M and p42-M.2 nucleic acids.

A p42, as previously described herein, “recombinant protein” is aprotein made using recombinant techniques, i.e. through the expressionof a recombinant nucleic acid as depicted above. A recombinant proteinis distinguished from naturally occurring protein by at least one ormore characteristics. For example, the protein may be isolated orpurified away from some or all of the proteins and compounds with whichit is normally associated in its wild type host, and thus may besubstantially pure. For example, an isolated protein is unaccompanied byat least some of the material with which it is normally associated inits natural state, preferably constituting at least about 0.5%, morepreferably at least about 5% by weight of the total protein in a givensample. A substantially pure protein comprises at least about 75% byweight of the total protein, with at least about 80% being preferred,and at least about 90% being particularly preferred. The definitionincludes the production of a protein from one organism in a differentorganism or host cell. Alternatively, the protein may be made at asignificantly higher concentration than is normally seen, through theuse of an inducible promoter or high expression promoter, such that theprotein is made at increased concentration levels. Alternatively, theprotein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertionsand/or deletions, as discussed below.

Included in the definition of p42 polypeptides are p42 polypeptidevariants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a p42 polypeptide, using cassette or PCR mutagenesis,scanning mutagenesis, gene shuffling or other techniques well known inthe art, to produce DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture as outlined above. However, variantp42 polypeptide fragments having up to about 100-150 residues may beprepared by in vitro synthesis using established techniques. Amino acidsequence variants are characterized by the predetermined nature of thevariation, a feature that sets them apart from naturally occurringallelic or interspecies variation of the p42 polypeptide amino acidsequence. The variants typically exhibit the same qualitative biologicalactivity as the naturally occurring analogue, although variants can alsobe selected which have modified characteristics as will be more fullyoutlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed p42 polypeptide variants arescreened for the optimal combination of desired activity. Techniques formaking mutations at predetermined sites in DNA having a known sequenceare well known. For example, the variations can be made usingoligonucleotide-mediated site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)], all of which are expressly incorporated byreference, PCR mutagenesis, or other known techniques can be performedon the cloned DNA to produce the p42 polypeptide variant DNA. Scanningamino acid analysis can also be employed to identify one or more aminoacids along a contiguous sequence. Among the preferred scanning aminoacids are relatively small, neutral amino acids. Such amino acidsinclude alanine, glycine, serine, and cysteine. Alanine is typically apreferred scanning amino acid among this group because it eliminates theside-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the variant [Cunningham and Wells, Science,244: 1081-1085 (1989), which is expressly incorporated by reference].Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976), which are expressly incorporated byreference]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used. Screening of the mutants orvariants is done using assays of p42 polypeptide activities and/orproperties as described herein.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the p42 polypeptide aredesired, substitutions are generally made in accordance with thefollowing chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the p42 polypeptide as needed, for example, toincrease the immunogenicity of the p42 polypeptide. Alternatively, thevariant may be designed such that the biological activity of the p42polypeptide is not altered. For example, glycosylation sites may beadded, altered or removed. p42 polypeptide may be designed to addphosphorylation sites.

p42 polypeptide fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating p42 polypeptidefragments by enzymatic digestion, e.g., by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restrictionenzymes, expressing the digested DNA and isolating the desired fragment.Yet another suitable technique involves isolating and amplifying a DNAfragment encoding a desired polypeptide fragment, by polymerase chainreaction (PCR). Oligonucleotides that define the desired termini of theDNA fragment are employed at the 5′ and 3′ primers in the PCR.Preferably, p42 polypeptide fragments share at least one biologicaland/or immunological activity with the native polypeptides shown in thefigures.

In another aspect of the invention, as noted above, we have found thatwhen a p42 amino acid sequence is expressed in insect cells, a pureproduct can be obtained without degradation or cleavage if DNA codingfor a MAD allele sequence is employed. An amino acid sequence of the p42antigen is considered to be of the MAD allele if it corresponds to theparts of gp195 of the MAD Plasmodium falciparum isolate which aredimorphic as compared with the amino acid sequences of the Wellcome andK1 isolates (Tanabe et al. 1987. J. Mol. Biol. 195:273). For example,gp195 of the FUP isolate is of the MAD allele (Chang et al. 1989. Proc.Natl. Acad. Sci. USA 86:6343; Chang et al. 1988. Exp. Parasitol. 67:1)(FUP isolate p42 amino acid sequence is as well).

The method for producing BVp42 according to this aspect of theinvention-involves infecting an insect cell host with a recombinantbaculovirus vector, that vector containing DNA coding for a p42 aminoacid sequence of surface protein gp 195 of the Plasmodium falciparum MADallele, operably linked to a baculovirus polyhedron promoter. Productionof BVp42 with the sequence of the MAD allele, preferably of the FUPisolate sequence, results in pure, undegraded antigen.

A high level of inhibition of parasite growth is exhibited by anti-BVp42sera. As noted above, strong inhibition of parasite growth by sera ofmonkeys vaccinated with Mab 5.2 affinity purified parasite gp195correlates with the ability to induce substantially complete protectionagainst infection. Other types of purification systems also can be usedas known in the art, such as, molecular exclusion chromatography, phenylHIC. Affinity chromatography also may employ a specific sequence ormoiety either native or added to p42 wherein the sequence or moietyspecifically binds to a compound or chemical such as in Ni-NTAchromatography, or an epitope can be added or fused to the p42 proteinwhich is purifed by affinity chromatography with an antibody the bindsto the added epitoped.

Without wishing to be bound by any theory of the invention, our resultsprovide new insights into the interpretation of the vaccination studydiscussed above utilizing parasite gp 195 that was affinity purifiedusing Mab 5.2. As shown herein, utilization of this Mab (now found by usto be specific for a conformational epitope located within the p42processing fragment) for affinity purification of parasite gp 195results in an antigen preparation that contains the gp195 precursor butthat is also highly enriched for several C-terminal processingfragments, including p42. Based on the analysis of the specificity ofanti-parasite gp195 antibodies utilizing the BVp42 polypeptide, amajority of antibodies produced by immunization with Mab 5.2 affinitypurified parasite gp195 is specific for the C-terminal p42 processingfragment. The importance of p42 epitopes in immunity is consistent withthe strong inhibition of parasite growth obtained with anti-BVp42 sera.There have been reports of several Mabs specific for this region whichinhibit in vitro parasite growth (Pirson et al. 1985. J. Immunol.134:1946; Blackman et al. 1990. J. Exp. Med. 172:379); Thus, it ispossible that the exceptional level of protection achieved in theprevious vaccination study was due to the focusing of the immuneresponse on C-terminal epitopes which appear to serve as targets offunctional effects such as the direct inhibition of parasite growth.

A study of the influence of MHC genes on immunological responsiveness togp195 had found that a variety of congenic mouse strains were capable ofproducing antibodies against gp195 (Chang et al. 1989. Proc. Natl. Acad.Sci. USA 86:6343). We have detected BVp42-specific antibodies in sevencongenic mouse strains immunized with purified, parasite gp195,indicating that individuals of many H-2 haplotypes are capable ofrecognizing epitopes within the smaller, p42 region of gp195. Thus,BVp42 can likely be used as a vaccine antigen in hosts of diversegenetic makeup.

Techniques known to one skilled in the art for expressing foreign genesin insect host cells can be used to practice the invention. Methodologyfor expressing polypeptides in insect cells is described, for example,in Summers and Smith. 1986. A Manual of Methods for Baculovirus Vectorsand Insect Culture Procedures, Texas Agricultural Experimental StationBull. No. 7555, College Station, Tex.; Luckow. 1991. In Prokop et al.,Cloning and Expression of Heterologous Genes in Insect Cells withBaculovirus Vectors' Recombinant DNA Technology and Applications,97-152; and in U.S. Pat. No. 4,745,051, all hereby incorporated byreference in their entirety. The techniques are summarized below.

To make BVp42, or an effective immunogenic part thereof, a polyhedronshuttle vector is used to shuttle all or part of the p42 coding sequenceinto a nuclear polyhedrosis virus. This vector contains the promotor ofthe polyhedron gene of a nuclear polyhedrosis virus and an availablecloning site for the insertion of a selected gene such that the selectedgene is under transcriptional control of the polyhedron promotor. Thus,the shuttle vector is engineered to contain the p42 sequence operablylinked to a baculovirus polyhedrin promoter.

For example, the p42 sequence can be inserted into a commonly usedpolyhedron transfer vector, such as pAC373, to produce a recombinantshuttle vector for introducing foreign genes into a nuclear polyhedrosisviruses, such as Polyhedron californica nuclear polyhedrosis virus(AcMNPV), resulting in a recombinant viral expression vector capable ofexpressing the gene encoding for p42 in a host insect cell. pAC373contains a deletion of the sequence between −8 (8 bases upstream fromthe polyhedron ATG and approximately 40 bases downstream from thepolyhedron transcriptional start site) and the natural BamHI sites atnucleotide+171 (Luckow et al. 1988. Trends in the Development ofBaculovirus Expression Vectors, Biotechnology, 6:47) resulting in aconstruct which can be used to express full-length gene which containsan internal ATG (N-formyl methionine initiation).

Any p42 coding DNA can be used to make the expression vector. Whilenatural DNA sequences of various Plasmodium falciparum isolates aredescribed herein, it is within ordinary skill in the art to vary thosesequences. Thus, non-natural DNA sequences, different from theparticular ones listed herein, can be used effectively in practicing theinvention. For example, given the degeneracy of the genetic code DNAsequences encoding a protein can be modified to optimize expression forparticular organisms and/or cells types, such as, tailoring the codonusage to those codons that are preferred by a given expression system,without modifying the desired amino acid sequence of the expressedprotein.

Similarly, non-natural amino acid sequences can be coded for by the DNAused in the transfer vector in order to obtain functional products thatobtain the advantages of the invention.

The C-terminus of BVp42 is preferably truncated, as discussed above, toremove the hydrophobic tail sequence (i.e., membrane anchor) to allowfor protein secretion. For example, the entire anchor sequence e.g.amino acids 1708-1725 of the FUP isolate and corresponding amino acidsof other isolates, can be deleted. An embodiment discussed in theexamples below deletes an additional two amino acids at the N-terminusof the anchor sequence.

Thus it is within the scope of the invention to delete portions of thep42 amino acid sequence which do not affect the beneficial resultobtained with the BVp42 products exemplified herein.

Transfer of the hybrid DNA to an expression vector is accomplished bytransfection of the host insect cell, e.g. Spodoptera frugiperda (sf)cells, with a mixture of both the recombinant transfer plasmid DNA andwild type nuclear polyhedrosis virus (MNPV).

The transfected plasmid DNA recombines with the homologous sequences inthe wild-type baculovirus genome to produce a viral genome that carriesan integrated copy of the foreign gene.

The recombinant expression vector, comprising the hybrid polyhedron-p42gene incorporated in the MNPV genome is then selected from the mixtureof nonrecombinant and recombinant baculoviruses. For example, thesupernatant containing the mixture of wild-type and recombinant buddedviruses is collected, clarified by centrifugation and used forsubsequent plaque assays. The preferred means of selection is by visualscreening for the absence of viral occlusion bodies or by plaquehybridization with nucleic acid coding for p42, such as described inKiefer et al. 1991. Growth Factors 5:115-127.

Expression of the p42 gene is accomplished by infecting susceptible hostinsect cells, such as sf cells, with the recombinant baculovirusp42-expression vector in an appropriate medium for growth. Propagationof the baculovirus p42-expression vector is achieved in the insect cellsthrough replication and assembly of infectious virus particles.

Various nuclear polyhedrosis viruses can be employed in makingexpression vectors for use in the invention. Suitable viruses include,but are not limited to, Polyhedron californica nuclear polyhedrosisvirus (AcMNPV), Spodoptera frugiperda nuclear polyhedrosis virus,Choristoneura fumiferana nuclear polyhedrosis virus, Spodopteralittoralis nuclear polyhedrosis virus, or Trichoplusia ni nuclearpolyhedrosis virus, all known in the art and commonly available.

Suitable insect host cells for use in the invention include, but are notlimited to, Sf9 (Spodoptera frugiperda), Spodoptera exiaua,Choristoneura fumiferana, Trichoplusia ni, and Spodoptera littoralis,Drosophila and other cells known in the art.

The polypeptide may be produced as either a fusion product, such as aheterologous protein containing part baculovirus amino acid sequencefused to Plasmodium falciparum sequence, or the product may merelycontain a Plasmodium falciparum sequence. Methods of making both typesof proteins are well known to one skilled in the art.

Accordingly, in a preferred embodiment, the invention provides fusionpolypeptides comprising a, first, p42 polypeptide and at least a secondpolypeptide, wherein the second polypeptide is a p42 polypeptide, asdefined herein, or a heterologous polypeptide. The p42 polypeptide andsecond polypeptide are fused covalently or non-covalently, withcovalently being preferred. As will be appreciated by those in the art,the fusion polypeptides of the invention can be configured in a varietyof ways. In one embodiment, the p42 polypeptide is fused to the carboxyterminus of the second polypeptide. Alternatively, the p42 polypeptideis fused to the amino-terminus of the second polypeptide. In anotheralternative embodiment, the p42 polypeptide is at a position internal tothe carboxy and amino termini or the second polypeptide. Accordingly,the p42 polypeptide and the second polypeptide are joined to producelinear fusions or branched fusions in any manner as the biology andactivity permits, although in general, N- or C-terminal fusions arepreferred to internal fusions.

In general, the fusion polypeptides of the invention can be made eitherrecombinantly or synthetically.

In a preferred embodiment, the p42 and second polypeptides are attachedthrough the use of functional groups on each that can then be used forattachment. Preferred functional groups for attachment are amino groups,carboxy groups, oxo groups and thiol groups. These functional groups canthen be attached, either directly or indirectly through the use of alinker. Linkers are well known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred linkers include, but arenot limited to, alkyl groups (including substituted alkyl groups andalkyl groups containing heteroatom moieties), with short alkyl groups,esters, amide, amine, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C2 alkene being especiallypreferred. Suitable crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimideesters, for example, esters with 4-azidosalicylic acid, homobifunctionalimidoesters, including disuccinimidyl esters such as3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such asbis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate. Accordingly, at least one amino acid that reactswith a crosslinking agent may be added to a p42 polypeptide or thesecond polypeptide antibody by insertion and/or substitution tofacilitate crosslinking.

In a preferred embodiment, the p42 and second polypeptides arecrosslinked to a third molecule, which accordingly provides a scaffoldfor linking the p42 and second polypeptides. Suitable scaffolds include,for example, peptides, carbohydrates, nucleic acids, lipids, smallorganic molecules and the like. The crosslinkers function to join thep42 and second polypeptide and preferably allow each component tofunction without interference from the other component or thecrosslinker.

In one embodiment, linear fusions are preferred. Accordingly, the p42polypeptide is directly fused either to the amino terminus, carboxyterminus, and/or is internal to the termini of the second polypeptide.In a preferred embodiment, linkers, spacers, or adapters comprised ofamino acids are used to join the p42 polypeptide and second polypeptide.In some embodiments, the fusion nucleic acid optionally encodes linkers,crosslinkers, spacers, or adapters, as needed. The number of amino acidscomprising the linker can be determined by routine experimentation by askilled artisan. The linkers comprising a sufficient number of aminoacids such that the p42 and second polypeptides function withoutinterference from the other. Accordingly, amino acids that comprise thelinker preferably do not substantially alter biological activity of thep42 or second polypeptide.

For example, useful linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO:77) and (GGGS)_(n) (SEQ ID NO:78), where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers such as the tether for the shaker potassiumchannel, and a large variety of other flexible linkers, as will beappreciated by those in the art. Glycine and glycine-serine polymers arepreferred since both of these amino acids are relatively unstructured,and therefore may be able to serve as a neutral tether betweencomponents. Glycine polymers are the most preferred as glycine accessessignificantly more phi-psi space than even alanine, and is much lessrestricted than residues with longer side chains (see Scheraga, Rev.Computational Chem. III73-142 (1992), expressly incorporated byreference). Secondly, serine is hydrophilic and therefore able tosolubilize what could be a globular glycine chain. Third, similar chainshave been shown to be effective in joining subunits of recombinantproteins such as single chain antibodies.

In a preferred embodiment, either or both p42 and the second polypeptideof the invention can comprise additional components or may be modifiedin other ways. For example, modification of the fusion polypeptidesinclude deamidation of glutaminyl and asparaginyl residues to thecorresponding glutamyl and aspartyl residues, respectively,hydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “amino groups oflysine, arginine, and histidine side chains [T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983), expressly incorporated by reference], acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the p42 and second polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence of the fusionpolypeptide components, and/or adding one or more glycosylation sitesthat are not present in the native sequences.

Addition of glycosylation sites may be accomplished by altering theamino acid sequence thereof. The alteration may be made, for example, bythe addition of, or substitution by, one or more serine or threonineresidues to the fusion polypeptide sequence (for O-linked glycosylationsites). The alteration also may be made, for example, by the additionof, or substitution by one or more Asn-Xaa-Ser/Thr sites (Xaa=any aminoacid; for N-linked glycosylation sites) in the fusion polypeptidesequence. The fusion polypeptide amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the p42 polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thefusion polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981), all of which are expresslyincorporated by reference.

Removal of carbohydrate moieties present on p42 or the fusionpolypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981), all of which are expressly incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987), expresslyincorporated by reference.

Another type of covalent modification of p42 or the fusion polypeptidecomprises linking the p42 or fusion polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, allof which are expressly incorporated by reference.

In one embodiment, the p42 polypeptide is fused to an epitope tag whichprovides an epitope to which an anti-tag antibody can selectively bind.The epitope tag is generally placed at the amino-or carboxyl-terminus ofthe p42 polypeptide but may be incorporated as an internal insertion orsubstitution as the biological activity permit. The presence of suchepitope-tagged forms of a p42 polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the p42 polypeptide to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988), which is expressly incorporated by reference]; the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,Molecular and Cellular Biology, 5:3610-3616 (1985), which is expresslyincorporated by reference]; and the Herpes Simplex virus glycoprotein D(gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990), which is expressly incorporated by reference].Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988), which is expressly incorporated byreference]; the KT3 epitope peptide [Martin et al., Science, 255:192-194(1992), which is expressly incorporated by reference]; tubulin epitopepeptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; andthe T7 gene protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393-6397 (1990), which is expressly incorporated byreference] and the histidine tag and metal binding sites (Smith, Ann.NY. Acad. Sci., 646:315-321 (1991), which is expressly incorporated byreference], with the Flag and histidine tag being preferred.

In a preferred embodiment, nucleic acid encoding a leader sequence canbe included in the expression vector and fused to the amino terminus ofthe p42 polypeptide to facilitate sorting through the endoplasmicreticulum and proper folding of the polypeptide product. For example,flg5 cDNA encoding a leader sequence can be inserted 5′ to the p42coding region using conventional techniques. The flg5 leader isdescribed in Kiefer et al. 1991.

Growth Factors 5:115-127. It is also disclosed in the Genebank and EMBLdata bases (accession no. M60485). In addition, the flg5 DNA sequenceused in a construct described in the examples below is shown in FIG. 7.The final purified product according to the invention may include aleader sequence, or the sequence may be cleaved during expression.Amino-terminal amino acid sequencing indicates that the polypeptide isappropriately cleaved.

The many variations in techniques for expressing and isolating BVp42will be apparent to one skilled in the art. For example, promoters knownin the art can be modified to alter expression. In addition, the numberand composition of the nucleotides in the region between the promoterand start of the open reading frame can be modified to alter expression.

The BVp42 product can be conventionally purified, such as, in part, byaffinity chromatography with a Mab specific for natural p42. Mab 5.2 isone such antibody. In another embodiment, BVp42 can be purified, forexample, by the addition of a tag to the p42 polypeptide as describedherein, preferably at the carboxy terminus, and purified by theappropriate affinity purification methods.

The BVp42 obtained is a variant of naturally occurring p42 that resultsfrom characteristic post-translational processing occurring in insectcells, especially Sf9 cells. Potential sites for post-translationalmodifications are shown in FIG. 6.

BVp42 has been found to be highly immunogenic in rabbits. High antibodytiters against the immunogen can be obtained which meet or exceed titersof animals immunized with purified parasite gp195. ELISA titers werefound similar in assays utilizing plates coated with either purified,parasite gp 195 or BVp42. More importantly, high titers were obtainedwhen anti-BVp42 antibodies were reacted with purified, parasite gp195 inan ELISA and in an indirect immunofluorescence assay with schizonts andmerozoites. IFA titers obtained after the fourth immunization with BVp42reached levels exceeding those obtained by immunization with purified,parasite gp195.

Yeast produced p42 (Yp42) consisting of the same p42 sequence (aminoacids nos. 1333 to 1705 in the FUP isolate) was found to be lessimmunogenic than BVp42, inducing lower antibody titers against theimmunogen. In addition, the cross-reactivity of anti-Yp42 antibodieswith parasite gp195 in the ELISA was much lower than cross-reactivity ofanti-BVp42 antibodies. Yp42 also induced much lower IFA titers thanBVp42, and statistically insignificant levels of parasite inhibition.

Immunoblotting studies demonstrated that most of the anti-BVp42 andanti-parasite gp 195 antibodies produced are specific fordisulfide-dependent, conformational epitopes present on the same set ofgp 195 processing fragments.

Accordingly, once made and purified, if necessary, the p42 polypeptidesand p42 polypeptide fusions are useful in a number of applications, forexample, in the induction of an anti-plasmodium immune response ortreatment of a disease state in a patient or individual. The p42polypeptides and fusions thereof of the present invention can be usealone or in combination with other therapeutic agents or carriers. In apreferred embodiment, the p42 polypeptides are employed as a vaccine.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

By “patient” or “individual” herein refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents or carriers includes simultaneous (concurrent) and consecutiveadministration in any order.

In a preferred embodiment, an adjuvant should be included with the BVp42in the vaccine of the invention in order to enhance the immune response.Such adjuvants include Freund's complete adjuvant, B30-MDP, LA-15-PH,(Hui et al. 1990. Vaccine. Cold Spring Harbor Press. 477-483; Hui et al.1991. Infection and Immunity 59:1585-1591; Hui et al. 1991 J. Immunol.147:3935-3941), Freund's incomplete adjuvant, saponin, aluminumhydroxide, MF59, MTP-PE, QA-21, ISA51 or other available adjuvants oradjuvant combinations. Freund's complete adjuvant is not generally usedclinically for human vaccines.

In a preferred embodiment, a pharmaceutically acceptable carrier isincluded in the therapeutic formulation, preferably a vaccine. Suchcarriers are well known to one skilled in the art and can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions. Therapeutic formulations are prepared for storage bymixing the active ingredient having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980),which is expressly incorporated by reference), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intramuscular,intraarterial or intralesional routes, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96, which is expressly incorporated byreference.

When in vivo administration of a fusion polypeptide is employed, normaldosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammalbody weight or more per day, preferably about 1 μg/kg/day to 10mg/kg/day, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or5,225,212, which are expressly incorporated by reference. It isanticipated that different formulations will be effective for differenttreatment compounds and different disorders, that administrationtargeting one organ or tissue, for example, may necessitate delivery ina manner different from that to another organ or tissue.

Where sustained-release administration of a polypeptide is desired in aformulation with release characteristics suitable for the treatment ofany disease or disorder requiring administration of the polypeptide,microencapsulation of the polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN—), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010, all of which are expressly incorporated by reference.

The sustained-release formulations of polypeptides were developed usingpoly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibilityand wide range of biodegradable properties. The degradation products ofPLGA, lactic and glycolic acids, can be cleared quickly within the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition.Lewis, “Controlled release of bioactive agents from lactide/glycolidepolymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers asDrug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41, whichis expressly incorporated by reference.

The vaccine preferably contains from about 0.1 to 5 mg in about a 0.1 mlto about 1.0 ml dose of BVp42. The vaccine can be administered in one ormore doses, the amount administered being adjusted to correspond withthe number of inoculations, either together or over a period of time.Administration can be carried out conventionally, preferablyparenterally.

In a preferred embodiment, the vaccine preferably induces an immuneresponse that substantially reduces the symptoms and manifestationsassociated with plasmodium infection, such as, the onset, the severity,and/or the duration of illness. The symptoms and manifestations include,for example, parasitemia, fever and chills which begin irregularly andlater become synchronous in a non-immune individual. In a most preferredembodiment the immune response prevents overt or clinical disease.

In a preferred embodiment, the vaccine substantially delays the onset ofa symptom in comparison to a non-vaccinated individual followinginfection at a dose that occurs normally in nature. Preferably, thesymptom is parasitemia. Accordingly, the vaccine delays the onset of asymptom by about 2 days, preferably reduces onset of a symptom by about4 days, more preferably by about 8 days, even more preferably by about16 days or higher.

In a preferred embodiment, the vaccine substantially reduces parasitemiain comparison to a non-vaccinated individual following infection at adose that occurs normally in nature. Accordingly, the vaccine reducesparasitemia by about 2 fold, preferably reduces parasitemia by about 10fold, more preferably by about 100 fold, even more preferably by about1,000 fold. In an even more preferred embodiment, parasitemia is reducedby 10,000 fold or higher and parasites can not be detected.

The present invention is further described in the examples below. Theexamples are for illustration purposes, and are not intended to limitthe scope of the invention. All patents, patent applications, andpublications and references cited therein are hereby expresslyincorporated by reference in their entirety.

EXAMPLES Example 1 Production and Purification of Baculovirus p42(BVp42)

The Uganda Palo Alto (FUP) Plasmodium falciparum p42 coding region fromAla₁₃₃₃ to Ser₁₇₀₅, was cloned into a Polyhedron californica nuclearpolyhedrosis virus (AcMNPV) polyhedrin promoter regulated expressionsystem (Luckow. 1988. Biotechnology 6:47).

FIG. 1 shows, schematically, the sequences that were cloned into a BVp42encoding shuttle vector. The coding sequence, shown at top, included theflg5 leader (Flg_(L), solid box) fused to the FUP isolate p42 codingregion from Ala₁₃₃₃ to Ser₁₇₀₅. The BamHI fragment finally obtained wascloned into a AcMNPV transfer plasmid, shown at the bottom of thefigure. The leader sequence for flg5 has been shown to direct secretionof the mature flg5 protein (Kiefer et al. 1991. Growth Factors5:115-127). When fused to p42 coding DNA in the baculovirus expressionsystem, the mature protein is also found to be secreted from the insectcells. “RV” is an EcoRV restriction site. The construct was made asfollows.

Oligomers encoding the flg5 leader (Kiefer et al. supra), 5′ portion ofFUP isolate p42 from Ala₁₃₃₃ to the HindIII site at amino acid Arg₁₃₆₂and BamHI sites were made on an automated DNA synthesizer (AppliedBiosystems, Foster City, Calif.), kinased, annealed, and ligated. Theoverlapping oligomers consisted of six 43-mers, two 46-mers and two19-mers. After digestion with BamHI, the ligation product was ligatedinto BamHI-digested and phosphatased pAB 114, a HindIII/SalI deletion ofpBR322 containing a BamHI linker (Barr et al 1988. J. Biol. Chem.263:16471). The HindIII/SalI fragment of p42 from a pAB 125 ADH₂-GAPDHalpha-factor construct (Hui et al. 1991. J. Immunol. 147:3935-3941),which includes an in-frame stop codon following Ser₁₇₀₅, was ligatedin-frame into this pAB114 vector to yield a flg5 leader fused tofull-length p42 flanked by unique BamHI sites. The BamHI fragment wassubsequently cloned into the BamHI site of the AcMNPV transfer plasmidpAc373 (Luckow. 1988. Biotechnology 6:47). The recombinant shuttlevector obtained was then used to transfer the coding sequence intoAcMNPV by homologous recombination through transfection of sf9 cells(Summers et al. 1986. Tex. Agr. Exp. Station Bull. No. 7555, CollegeStation, Tex.). Recombinant viruses were initially identified visuallyby occlusion negative phenotype and plaque hybridization with the BamHIfragment labeled by the oligomer primed extension method (Feinberg etal. 1984. Anal. Biochem. 137:266). The recombinant AcMNPV expressionvector obtained, Flg5LFUP42AcNPV, was deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA 20852, onJan. 28, 1992 and has been assigned accession number VR2354. Infectionof sf9 cells was carried out conventionally per Summers et al. 1986.supra.

Protein expression was initially monitored in roller bottles. Productionwas then scaled up to 0.5 liter batches in 2.8 liter Fembach shakerflasks. Culture supernatants were concentrated 10 to 50-fold bytangential flow ultrafiltration (Amicon). Recombinant BVp42 was purifiedfrom the concentrated supernatant by the affinity chromatographytechnique utilized to purify parasite gp195 as described by Siddiqui etal. 1987.

FIG. 2A shows silver stains (lane 1) and immunoblots (lane 2) of theaffinity chromatography purified BVp42. The silver stained antigens wereelectrophoresed in SDS polyacrylamide gels. The immunoblots were reactedwith a rabbit anti-parasite gp195 serum pool.

A single band migrating at approximately 44 kDa was observed in thesilver stain. In the immunoblots, the purified BVp42 displayed majorimmunoreactive species at the positions of the major protein bands,accompanied by minor reactivities with proteins of higher and lowermolecular weight.

The DNA sequence of the BVp42 construct from BamHI to SalI is shown inFIG. 7, the numbering starting with the first nucleotide of BamHI andending with the last nucleotide of SalI. A restriction map is shownschematically at the top of the figure and the same sites are shownadjacent to the corresponding sequences below.

Edman sequence of the expressed protein of BVp42 resulted in a mainN-terminal sequence consistent with the expected p42 N-terminus:ISVTMDNILS (SEQ ID NO:9) The yield of this sequence was lower thanexpected, suggesting that some of the material may have a blockedN-terminus. A minor sequence corresponding to the N-terminus of bovineserum albumin (BSA) was also detected, indicating that there was a traceamount of BSA in the preparation.

Example 2 Production and Purification of Modified p42 Nucleic AcidConstructs (p42-M and p42-M.2)

The original p42 nucleotide sequence (SEQ ID NO: 1) was substantiallymodified in order to optimize recombinant p42 polypeptide expression,achieve higher recombinant polypeptide concentrations of immunogen inculture supernatants, and to enhance the purity of recovered p42immunogen. The first embodiment of the modified p42 nucleotide sequence(p42-M) comprises nucleotides 1-1232 of SEQ ID NO: 6 (FIG. 12) andincludes 18 nucleotides which encode a histidine tail. The deduced aminoacid sequence for p42-M (p42-M polypeptide) comprises amino acids 1-402of SEQ ID NO: 16. The second embodiment of the modified p42 nucleotidesequence (p42-M.2) comprises nucleotides 1-1200 of SEQ ID NO: 17 (FIG.12) and excludes the 18 nucleotides encoding the histidine tail. Thededuced amino acid sequence for p42-M.2 (p42-M.2 polypeptide) comprisesamino acids 1-392 of SEQ ID NO: 18.

The strategy for production of the p42-M (SEQ ID NO: 6) and p42-M.2 (SEQID NO: 17) constructs was implemented in order to alter the codon usagein the p42 gene for compatibility with the codon usage of genes of theTrichoplusia ni (HIGH FIVE™) host cells used for p42 polypeptideexpression. The following steps were involved in this strategy: (a) acodon usage table (FIG. 27) was obtained for Trichoplusia ni genes froma public data base (e.g., the codon usage database of Y. Nakamura,Kazusa DNA Research Institute; (b) based on the codon usage table, thenucleic acid sequence of p42 was altered to obtain the same frequencyfor each codon as a typical Trichoplusia ni gene. This modificationinvolved calculating the number of codons for each animo acid of the P.falciparum MSP-1 gene sequence within the p42 coding region, determiningthe codon usage for each amino acid in the p42 nucleic acid codingregion, and altering this codon usage frequency to be consistent withthe Trichoplusia ni usage. Once the actual and desired codon frequencieswere determined, appropriate codons were substituted over the entire p42coding region sequence to achieve the desired codon frequencies. Themodified sequence was then scanned for any stop codons that may havebeen inadvertently introduced as a result of these substitutions. Stopcodons which were identified were corrected. The promoter andtranslation initiation region were additionally modified to conform tooptimal sequences for gene expression (Ranjan, A., Hasnain, S.E., VirusGenes. 9(2): 149-153, 1995). Finally, conservative nucleotidesubstitutions were included in the construct to introduce severalconvenient restriction endonuclease cleavage sites throughout the genein order to facilitate genetic manipulations.

The p42-M (SEQ ID NO: 6) and p41-M.2 (SEQ ID NO: 17) nucleic acidsequences were further modified to contain an optimized promoter andtranslation initiation region (GGATCCACTAAAATGTGGTCTTGGAAG) based onRanjan and Hasnain, 1995. The translation initiation region was selectedby comparing codon usage of all the known gene sequences from Autographacalifornica nuclear polyhedrosis virus (AcNPV) with codon usage of thegene sequence for firefly luciferase (luc) and codon usage of the genesequence for the beta subunit of human chorionic gonadotropin (betahCG). The luc gene and beta hCG gene are expressed at different levelsin the baculovirus system. The highly expressed luc gene showed a codonusage similar to AcNPV genes, as reflected by a very low D-squaredstatistic value (0.78) and a similar G/C usage (45%) at wobblepositions. However, the underexpressed beta hCG gene displayed a highD-squared value (7.3) and G/C usage (82.5%) at the wobble base position.Alignment of the 20 nucleotides around the initiation codon of 23 AcNPVgenes identified a novel consensus translation initiation sequence ofaag/ta/tat/aa/cAAaATGaa/ct/ag/aAan (SEO ID NO:79). The most criticalfeature of this translation initiation region, the triple A stringimmediately before the initiation codon, was selected for use with thep42-M and p41-M.2 constructs.

The process for generating the final p42-M and p42-M.2 constructs usinga modified PCR gene assembly protocol (FIG. 21) with overlappingoligonucleotides (40- to 50-mers; Table 12) encoding the complete p42-M(SEQ ID NO: 6) and p42-M.2 (SEQ ID NO: 17) constructs was more complexthan originally contemplated and the various steps taken to generatethese constructs is described below. The complete sequences of the finalp42-M (SEQ ID NO: 6) and p42-M.2 (SEQ ID NO: 17) constructs are providedin FIG. 12.

Initial studies to determine feasibility of the protocol were carriedout with 20 forward and 20 reverse p42 MSP-1 oligonucleotide primers(provided by GIBCO Invitrogen Corp., Carlsbad, Calif.) encompassing thep42-M and p42-M.2 constructs under standard PCR conditions, using Taqpolymerase. These initial studies indicated it was not possible toreconstruct the entire p42-M/p42-M.2 construct in a single step usingeach of the 40 oligonucleotides (GIBCO Invitrogen Corp., Carlsbad,Calif.). In later experiments, a more viable strategy was employed inwhich pools of nucleotides corresponding to sequence blocks flanked byintroduced restriction endonuclease sites were assembled with theintention that several fragments would be generated which couldsubsequently be combined to form the complete p42-M and p42-M.2constructs.

Production of Construct I:

The strategy for production of the first construct generated isillustrated in FIG. 22 as Construct I. It was necessary to developunique conditions, described below, for the production of each fragmentsince it was often not possible to simply mix the overlappingnucleotides together and obtain the specific, desired PCR product.

Fragment I(a) was generated in two steps: (1) 3 sets of top and bottomstrand primers for the first half and 3 sets of top and bottom strandprimers for the second half of this fragment (GIBCO Invitrogen Corp.,Carlsbad, Calif.) were amplified separately by PCR (60 cycles), and (2)the combination of the products (215 bp and 277 bp, respectively) of thetwo reactions were combined with the top (forward) and bottom (reverse)strand primers (Oligonucleotides #1, #36; Table 12) and carried througha second PCR reaction (60 cycles) to generate the desired 350 bpfragment. Attempts were made to utilize a high fidelity DNA polymerasesuch as the Turbo Pfu DNA polymerase (Stratagene) or Expand HighFidelity DNA polymerase (Boehringer Manneheim) for this reaction,however a product was obtained only when Taq polymerase was used.

Fragment I(b) was generated in three steps by sequentially (1) poolingOligonucleotides #23 through #27 and #53 through #57 (Table 12) andcarrying out a PCR assembly reaction for 45 cycles, (2) taking theproduct of this first PCR assembly step, combining it with the forwardprimer (Oligonucleotide #23) and the reverse primer (Oligonucleotide#53) and performing an amplification reaction for 45 cycles, resultingin a 215 bp amplified product, and (3) digesting this amplified productwith EcoRI and SacI to generate the 170 bp Fragment I(b).

Fragment I(c) was the only fragment which could be generated by simplycombining all of the overlapping oligonucleotides (Oligonucleotides #9through 20 and #40 through 50; Table 12). Twelve top stand and twelvebottom strand oligonucleotides were mixed (Oligonucleotides #9 through20 and #40 through 50; Table 12) and elongated in the assembly stage ofthe reaction. The product of this reaction was subsequently diluted andcombined with the forward and reverse primers for the I(c) fragment(GIBCO Invitrogen Corp., Carlsbad, Calif.), amplified by PCR (60cycles), and purified. High fidelity enzyme Turbo Pfu DNA polymerase(Stratagene) was used to generate the I(c) fragment.

Each of the three amplified fragments were purified by the Genecleansilica matrix method (Bio 101/Qbiogene, Carlsbad, CA digested with theappropriate restriction enzymes to generate the restriction fragmentsshown in FIG. 22, and ligated with T4 DNA ligase. Since it was notpossible to obtain cloned inserts directly with the ligation product,the ligated mixture was reamplified by PCR (35 cycles) with end primers(GIBCO Invitrogen Corp., Carlsbad, CA) and a high fidelity DNApolymerase(EXPAND™, a combination of Taq and Pwo DNA polymerases). Thisamplified product was digested with the restriction enzymes BamHI andKpnI, and ligated to an appropriately digested pUC 18 vector to generatea number of clones with the appropriate 1.2 kb insert.

Several of the clones containing the correct insert were evaluated byautomated DNA sequencing. Several nucleotide errors were noted in thebeginning of the sequence within fragment I(a) and a few additionalpoint mutations were scattered in other portions of the gene. (FIG. 22,Construct I, location of mutations indicated by aserisks).

Production of Construct II:

The large number of sequence errors obtained in the beginning of thegene (FIG. 22) were thought to be due to amplification of this region inmultiple stages involving several amplification reactions, the largenumber of cycles in each amplification, and usage of the error-prone Taqpolymerase. Consequently, it was decided that this region of the genewould be reconstructed using a different approach in order to generatefewer mutations. The reconstruction included defining conditions underwhich it was possible to use the high fidelity Pfu polymerase(Stratagene) for PCR amplification of this region as well as fewercycles (40 vs. 50 cycles) for PCR reactions.

In the first step of this process, the 5′-terminal BamHI-AccI fragmentwas generated in a separate PCR assembly reaction using Oligonucleotides#1, #2, #3, #31, #32, and #33 (Table 12), and 45 cycles. The BamHI-AccIfragment was then ligated using DNA T4 ligase with the AccI-EcoRIfragment to produce fragment II(a). The 3′ end of this fragment wascreated by the assembly of overlapping oligonucleotides(Oligonucleotides #4 through #6 and #34 through #36; Table 12)corresponding to the 200 bp AccI-EcoRI fragment and production of asecondary amplification product by combining this AccI-EcoRI fragmentwith the 615 bp EcoRI-EcoRI internal fragment obtained by restrictiondigestion of Construct I in a PCR assembly reaction.

Since the rest of the gene in Construct I had been generated using highfidelity polymerases, it was likely that the few errors obtained in thisregion were due to contamination of the oligonucleotide primers witherror sequences. New primers of higher purity (HPLC-purifiedoligonucleotides) were used (GIBCO Invitrogen Corp., Carlsbad, Calif.)to encompass the HindIII-SalI region of fragment II(b). The HindIII-SalI180 bp fragment assembled from these oligonucleotides was then combinedwith the entire insert of Construct I (as a template) to produce thecomplete fragment II(b) using the 180 bp HindIII-SalI fragment as theforward primer and Oligonucleotide #50 (Table 12) as the reverse primer(GIBCO Invitrogen Corp., Carlsbad, Calif.).

Fragments II(a) and II(b) were then digested with the appropriaterestriction endonucleases. These fragments were purified by Genecleanand ligated with DNA T4 ligase to the pUC vector to generate ConstructII. Two clones (c1 and c2) containing the appropriate sized insert wereobtained of Construct II and both were sequenced. They, however, werefound to contain a few, new, mutations, as indicated in FIG. 22(Construct II, c1 and c2; location of mutations indicated by asterisks).

Production of Construct III:

It was noted that between the two clones (c1 and c2), the correctsequence of the complete gene could be generated by fragment reassemblywith the exception of the internal EcoRI-SacI region. Therefore, it wasdecided to resynthesize the EcoRI-SacI region using a new set ofoligonucleotides (GIBCO Invitrogen Corp., Carlsbad, Calif.) and torecombine this gene assembly product after restriction enzyme treatmentwith the appropriate restriction fragments for the other regions fromeither the c1 or the c2 clones (FIG. 2, Construct III). These fragmentswere inserted into pUC 18 using T4 DNA ligase and recombinant colonies(Epicurean Coli XLI-Blue cell line, Stratagene, La Jolla, Calif.)containing the appropriate insert were sequenced. At the same time, theEcoRI-SacI fragment was subcloned into pUC and sequenced. The sequencingresults indicated a single deletion within the EcoRI-SacI region in boththe complete gene construct and the construct containing only theEcoRI-SacI region (FIG. 22, Construct III; location of mutationindicated by asterisk).

Production of Construct IV:

Since only a single mutation remained in the complete p42-M/p42-M.2construct, this error was repaired using oligonucleotide-directed sitespecific mutagenesis. To accomplish this, a complementary primer pair(Oligonucleotides #29 and 59; Table 12), provided by GIBCO InvitrogenCorp., Carlsbad, Calif. was designed which encompassed the deletion andmet appropriate criteria for site-directed mutagenesis (GC content,length, T_(m)). In order to avoid further problems with mutations,highly purified oligonucleotides prepared by polyacrylamide gelelectrophoresis were used (GIBCO Invitrogen Corp., Carlsbad, Calif.).This oligonucleotide pair was used to prime synthesis of the entirerecombinant plasmid using a minimal number of PCR cycles (15 cycles) andthe high fidelity Turbo Pfu DNA polymerase (Stragene) which createsblunt-ended PCR products. The double stranded product was treated withDpnI to remove any parental template and the blunt-end product wascircularized using T4 DNA ligase. This product was then used totransform E. coli to generate clones containing Construct IV. ConstructIV was sequenced and the results indicated the correct DNA sequence overthe entire gene. The insert of this plasmid was purified and subclonedinto the pAC 373 transfer vector in order to generate a recombinantbaculoviris containing the final form of the modified, optimized p42-M(or p42-M.2) baculovirus_construct.

The modified p42 baculovirus, now referred to as BVp42-M (modified BVp42of the MAD20 allele) was plaque-purified and studied in small-scale(shaker flask) expression experiments. A comparison of p42 and p42-Mpolypeptide expression between parallel cultures infected with eitherthe original BVp42 construct or the new BVp42-M construct is presentedin FIGS. 23A and 23B. In terms of the kinetics of expression, the p42-Mpolypeptide encoded by BVp42-M appears to be expressed much more rapidlythan the p42 polypeptide encoded by the BVp42 construct, and the p42-Mpolypeptide reaches peak expression levels relatively early (between 36and 48 hours after infection) as can be visualized by both the captureELISA results (FIG. 23A) and by Coomassie staining of SDS-Page (FIG.23B). This peak is rather sharp, and is followed by a rapid drop inp42-M polypeptide levels. This drop is most likely due to degradation bycellular proteases. The drop in p42-M polypeptide levels detected in thecapture ELISA assay at 54 h is accompanied by a disappearance of thep42-M polypeptide band in the corresponding Coomassie-stained gel. Themagnitude of peak expression also appears to be much higher for the newBVp42-M construct as compared to the original BVp42.

In this experiment, the peak p42 polypeptide concentration of theoriginal BVp42 construct was estimated at 0.7 μg/ml using theantigen-capture ELISA while the peak p42-M polypeptide concentration ofthe new BVp42-M construct was estimated at 2.3 μg/ml, approximately afour-fold increase. This is consistent with the fact that this is thefirst time that the p42-M band is detectable in Coomassie stains ofcrude supernatants of the infected culture. The enhanced level ofexpression of p42-M led to the development of the p42-M.2 construct(lacking the 18 nucleotides encoding the hexa-histidine tag). Theprojected higher yield for the p42-M.2 construct should enablepurification of the p42-M.2 polypeptide using alternativechromatographic techniques and the avoidance of the use of metal chelatechromatography.

It should be noted that the cultures were infected at an MOI which maynot be the optimal MOI for p42-M/p42-M.2 polypeptide expression by thisconstruct. The accelerated kinetics of p42-M/p42-M.2 expression providean advantage for protein recovery since it will be possible to harvestsupernatants while cell viability is still high and proteaseconcentrations are minimal. The kinetic studies are being refined todetermine the precise peak of expression, since there appears to be someproteolysis by 48 h, and the actual peak for p42-M/p42-M.2 is likely tooccur between 36 and 48 h.

A mAb affinity chromatography assay was carried out with 75 ml p42-M.2culture supernatant harvested at 40 h post-infection. A total of 500 μgof highly purified p42-M 0.2 protein was obtained, indicating an actualyield of 6.6 mg p42 per liter of culture. A Coomassie stained SDS-PAGEgel of this preparation is shown in FIG. 24. It is noted that thep42-M.2 band is the predominant band in this gel, with no indication ofdegradation or processing. In previous studies, an approximatelyten-fold increase in expression levels has been observed in thebioreactor (collaborative studies performed at Ares-Serono, Geneva) ascompared to shaker flasks. Consequently, it is projected that bioreactorexpression levels of the BVp42-M.2 construct will be approximately 70μg/ml or 70 mg/L. This level of expression would be more than adequatefor large scale production and for clinical trials of this malariaantigen. Thus, a 20 L culture would be projected to produce 1.4 g ofp42-M.2 protein.

Immunoreactivity of purified p42-M.2 polypeptide has been evaluatedusing several available mAbs specific for disulfide-dependent,conformational C-terminal determinants: Mabs 5.2 and AD9.1, Siddiqui etal., 1987. Proc Natl Acad Sci 84(9): 3014-3018; Locher et al., 1996. ExpParasitol 84(1): 74-83); mAb G13; as well as Dr. S. Longacres' G14 mAb,the kind gift of Dr. Shirley Longacre; and a rabbit polyclonal antiserumagainst p42, Chang et al., 1992. J Immunol 149(2):548-555. The p42-M.2polypeptide reacted strongly with each of these antibodies by immunoblot(FIG. 25).

Development of a Simplified, Non-Denaturing Purification Scheme forEnhancing Recovery and Purity of p42-M.2

The purification scheme of p42-M.2 outlined below is based on thepolypeptide's novel binding characteristics. Due to its amphipathicnature, p42-M.2 polypeptide displays a high affinity for hydrophobicligands attached to a chromatographic matrix. Preliminary methodscouting experiments demonstrated that one matrix exhibiting a highbinding interaction and selective elution properties for p42-M.2polypeptide was Phenyl Sepharose. The p42-M.2 polypeptide boundquantitatively and with high affinity to Phenyl Sepharose. Contaminantsbound with a lower affinity could be eluted with a low salt buffer.Subsequently, a partially-purified p42-M.2 polypeptide preparation wasobtained by elution with distilled water. This preparation was greatlyenriched for the p42-M.2 polypeptide but also contained high and lowmolecular weight contaminants (FIG. 26, lane 2).

The p42-M.2 polypeptide enriched preparation was further purified bychromatography using Cibachron Blue Sepaharose, a ligand which binds tokinases, dehydrogenases and most other enzymes requiringadenyl-containing cofactors (e.g., NAD+), coagulation factors,interferons, lipoproteins and albumin. The p42-M.2 polypeptide bindswith high affinity to Cibachron Blue and may be eluted only underhigh-stringency elution conditions (FIG. 26, lanes 7-8), enabling itsseparation from the remaining contaminants, some of which do not bind tothis ligand, or can be eluted under milder conditions (FIG. 26, lanes3-6). Consequently, a highly purified p42-M.2 polypeptide preparation isobtained in essentially a two-step purification procedure.

In addition to its relevance for purification, it is possible that theCibachron Blue binding properties to p42-M.2 polypeptide may haveimplications for anti-malarial drug development. If binding can be shownto be adenyl-cofactor specific, it is possible that structural analogsof this ligand may inhibit parasite invasion, growth, and/ordifferentiation. The nature of the binding interaction between p42-M.2polypeptide and Cibachron Blue is currently under investigation for drugdevelopment potential.

TABLE 12 Oligonucleotide Primers Used for the Production of the Modifiedp42 Nucleic Acid Sequence (p42-M and p42-M.2). FORWARD OLIGONUCLEOTIDEPRIMERS (5′→3′) Numbers (#s) 1-29 # 1 ATT GGA TCC ACT AAA ATG TGG TCTTGG AAG TGT CTT TTA TTC TGG GCT GT (F1) (SEQ ID NO: 19) # 2 CCA CTC TTTGCA CAG CAG CGA TCT CTG TTA CTA TGG ACA ACA TCC TCA GTG (F2) (SEQ ID NO:20) # 3 CGA GTA CGA CGT AAT CTA CCT AAA GCC CCT TGC CGG TGT CTA CCG TTCAT (F3) (SEQ ID NO: 21) # 4 GAA AAG AAT ATT TTC ACG TTC AAC CTC AAC CTAAAT GAC ATC CTC AAC TCG CG (F4) (SEQ ID NO: 22) # 5 CGA AAA TAC TTC CTCGAC GTG TTG GAA TCC GAC CTT ATG CAA TTT AAG CAC (F5) (SEQ ID NO: 23) # 6ACG AGT ACA TCA TAG AGG ACA GCT TCA AGC TCT TGA ATT CAG AAC AGA AGA ACA(F6) (SEQ ID NO: 24) # 7 GTC CTA CAA ATA CAT TAA GGA GTC TGT TGA GAA CGACAT CAA GTT CGC CCA GGA (F7) (SEQ ID NO: 25) # 8 TAC TAT GAG AAA GTC CTGGCT AAA TAC AAG GAC GAC TTG GAA AGC ATT AA (F8) (SEQ ID NO: 26) # 9 AAAGAA GAG AAG GAA AAG TTT CCG AGC TCT CCA CCC ACA ACT CCC CC (F9) (SEQ IDNO: 27) # 10 AAG ACC GAC GAG CAG AAA AAA GAA AGT AAG TTC CTT CCA TTC CTCAC (F10) (SEQ ID NO: 28) # 11 ACT CTA TAT AAC AAC CTG GTG AAC AAG ATTGAT GAC TAC TTA ATC AAC TTG AAG GCG (F11) (SEQ ID NO: 29) # 12 GAC TGTAAC GTC GAA AAG GAT GAA GCC CAC GTT AAG ATC ACC AAG CTT TCC (F12) (SEQID NO: 30) # 13 CCA TCG ACG ATA AGA TTG ACC TGT TTA AGA ACC ACA ACG ATTTCG ACG CA (F13) (SEQ ID NO: 31) # 14 TGA TCA ACG ACG ATA CTA AGA AAGACA TGC TTG GAA AAC TGG TGT CGA CAG (F14) (SEQ ID NO: 32) # 15 AAA CTTCCC GAA CAC CAT TAT AAG CAA GCT GAT CGA AGG AAA GTT TCA (F15) (SEQ IDNO: 33) # 16 AAC ATC TCT CAG CAT CAA TGC GTG AAG AAG CAA TGT CCC GAG AATTCA G (F16) (SEQ ID NO: 34) # 17 CCA CTT AGA CGA AAG GGA GGA ATG TAA ATGCCT GCT GAA TTA TAA ACA GG (F17) (SEQ ID NO: 35) # 18 GTG CGT AGA GAATCC TAA CCC AAC CTG TAA CGA AAA TAA CGG TGG CT (F18) (SEQ ID NO: 36) #19 CGC TAA GTG TAC CGA GGA GGA CAG CGG TTC CAA TGG CAA GAA AAT AAC T(F19) (SEQ ID NO: 37) # 20 GAA GCC CGA TAG TTA CCC TCT CTT CGA CGG TATCTT CTG CTC CCC ACC (F20) (SEQ ID NO: 38) # 21 TTC GAC GCA ATC AAA AAGTTG ATC AAC GAC GAT ACT AAG (F-14A) (SEQ ID NO: 39) # 22 AAA GAC ATG CTTGGA AAA CTG CTG TCG ACA GGC TTG GTC CA (F-14B) (SEQ ID NO: 40) # 23 GAGGAC AGC TTC AAG CTC TTG AAT TCA GAA CAG AAG AAC AC (F-6A) (SEQ ID NO:41) # 24 CCT CCT AAA GTC CTA CAA ATA CAT TAA GGA GTC TGT TGA G (F-7B)(SEQ ID NO: 42) # 25 AAC GAC ATC AAG TTC GCC CAG GAA GGA ATT AGC TAC TATG (F-7C) (SEQ ID NO: 43) # 26 AGA AAG TCC TGG CTA AAT ACA AGG ACG ACTTGG AAA GCA T (F-8D) (SEQ ID NO: 44) # 27 TAA GAA GGT AAT CAA AGA AGAGAA GGA AAA GTT TCC GAG (F-9E) (SEQ ID NO: 45) # 28 CTC TCC ACC CAC AACT (F-9F) (SEQ ID NO: 46) # 29 GGA ATT AGC TAC TAT GAG AAA GTC CTG GCTAAA TAC AAG G (F-7D) (SEQ ID NO: 47) REVERSE OLIGONUCLEOTIDE PRIMERS(5′→3′) Numbers (#S) 31-59 # 31 CTG CTG TGC AAA GAG TGG CGG TCA CCA AGACAG CCC AGA ATA AAA GAC A (R1) (SEQ ID NO: 48) # 32 AGG TAG ATT ACG TCGTAC TCG TTC TCG AAG CCA CTG AGG ATG TTG TCC ATA (R2) (SEQ ID NO: 49) #33 TTG AAC GTG AAA ATA TTC TTT TCT ATC TGT TTC TTC AAT GAA CGG TAG ACA(R3) (SEQ ID NO: 50) # 34 ACG TCG AGG AAG TAT TTT CGC TTG TTG AGG CGCGAG TTG AGG ATG TC (R4) (SEQ ID NO: 51) # 35 CTG TCC TCT ATG ATG TAC TCGTTA GAG CTA ATG TGC TTA AAT TGC ATA AGG TC (R5) (SEQ ID NO: 52) # 36 AGACTC CTT AAT GTA TTT GTA GGA CTT TAG GAG GGT GTT CTT CTG TTC TGA ATT CAA(R6) (SEQ ID NO: 53) # 37 GCC AGG ACT TTC TCA TAG TAG CTA ATT CCT TCCTGG GCG AAC TTG A (R7) (SEQ ID NO: 54) # 38 AAA CTT TTC CTT CTC TTC TTTGAT TAC CTT CTT AAT GCT TTC CAA GTC GTC (R8) (SEQ ID NO: 55) # 39 TTTCTG CTC GTC GGT CTT TGC AGG CGA TGG GGG AGT TGT GGG TG (R9) (SEQ ID NO:56) # 40 GTT CAC CAG GTT GTT ATA TAG AGT TTC GAT GTT GGT GAG GAA TGG AAGGAA CT (R10) (SEQ ID NO: 57) # 41 TCC TTT TCG ACG TTA CAG TCA TTA ATTTTC GCC TTC AAG TTG ATT AAG TAG T (R11) (SEQ ID NO: 58) # 42 GTC AAT CTTATC GTC GAT GGC TTT GAG ATC GGA AAG CTT GGT GAT CCT AAC (R12) (SEQ IDNO: 59) # 43 TTC TTA GTA TCG TCG TTG ATC AAC TTT TTG ATT GCG TCG AAA TCGTTG TGG T (R13) (SEQ ID NO: 60) # 44 ATG GTG TTC GGG AAG TTT TGG ACC AAGCCT GTC GAC AGC AGT TTT CC (R14) (SEQ ID NO: 61) # 45 CAT TGA TGC TGAGAG ATG TTC AGC ATA TCC TGA AAC TTT CCT TCG ATC AG (R15) (SEQ ID NO: 62)# 46 CTC CCT TTC GTC TAA GTG GCG GAA GCA ACC TGA ATT CTC GGG ACA TTG(R16) (SEQ ID NO: 63) # 47 GGT TAG GAT TCT CTA CGC ACT TGT CTC CTT CCTGTT TAT AAT TCA GCA GGC (R17) (SEQ ID NO: 64) # 48 CTC CTC GGT ACA CTTAGC GTC AGC ATC GCA GCC ACC GTT ATT TTC G (R18) (SEQ ID NO: 65) # 49 AGGGTA ACT ATC GGG CTT CGT GCA TTC GCA AGT TAT TTT CTT GCC ATT GG (R19)(SEQ ID NO: 66) # 50 GGC GTA GGT ACC TTA TTA ATG ATG ATG ATG ATG ATG AGGTGG GGA GCA GAA GAT AC (R20) (SEQ ID NO: 67) # 51 TTT TTG ATT GCG TCGAAA TCG TTG TGG (R-13A) (SEQ ID NO: 68) # 52 CAG TTT TCC AAG CAT GTC TTTCTT AGT ATC GTC GTT GAT CAA C (R-13B) (SEQ ID NO: 69) # 53 AGT TGT GGGTGG AGA GCT CGG AAA CTT TTC CTT CT (R-9A′) (SEQ ID NO: 70) # 54 CTT CTTTGA TTA CCT TCT TAA TGC TTT CCA AGT CGT CCT T (R-9B′) (SEQ ID NO: 71) #55 GTA TTT AGC CAG GAC TTT CTC ATA GTA GCT AAT TCC TTC CT (R-8C′) (SEQID NO: 72) # 56 GGG CGA ACT TGA TGT CGT TCT CAA GAG ACT CCT TAA TGT A(R-8D′) (SEQ ID NO: 73) # 57 TTT GTA GGA CTT TAG GAG GGT GTT CTT CTG TTCTGA ATT C (R-7E′) (SEQ ID NO: 74) # 58 AAG AGC TTG AAG CTG TCC TC(R-6F′) (SEQ ID NO: 75) # 59 CCT TGT ATT TAG CCA GGA CTT TCT CAT AGT AGCTAA TTC C (R-7D) (SEQ ID NO: 76)p42-M.2 Polypeptide Purification SchemePhenyl Sepharose Primary Purification Protocol:

Mix an equal volume of p42-M.2 polypeptide lysate with 3M ammoniumsulfate (pH 7.2). Mix gently. Centrifuge at 15K (4° C.; 30 minutes) toclear lysate of particulates. Lipids will float and form a layer on thetop. Carefully decant. Avoid lipid top layer if possible.

Prepare Phenyl Sepharose column. Pour slurry into column. Useperistaltic pump to add washes and eluants. Wash beads with 10 volumeswater, 10 volumes of 1.5 M ammonium sulfate in 20 mM NaH₂PO₄ (pH 7.2).Pass lysate over column.

Wash column with 10 volumes of 1.5 M Ammonium Sulfate in 20 mM 20 mMNaH₂PO₄ (pH 7.2).

Elute column with 5 volumes of 0.75M Ammonium Sulfate in 20 mM NaH₂PO₄(pH7.2). Follow with 5 volumes of 20 mM NaH₂PO₄ (pH 7.2) and 5 volumesof dH₂O.

Check fractions for p42-M.2 polypeptide by dot blot. The purest p42-M.2protein will be in water eluate.

Cibachron Blue Secondary Purification:

Pool fractions of Phenyl Sepharose purified p42-M.2 polypeptide. AddTween-80 for a final concentration of 0.5%.

Prepare column. Wash with 10 volumes water. Equilibrate column with 10volumes of 20 mM NaH₂PO₄ (pH 7.2) and 0.5% Tween-80.

Pass sample over column. Collect flow-thru to check if it has all bound.Wash column with 10 volumes of 20 mM NaH₂PO₄ (pH 7.2) and 0.5% Tween-80.

Elute column with 10 volumes of 2M NaCl in 20 mM NaH₂PO₄ (pH 7.2). Elutewith 10 volumes [50% Ethylene Glycol and 50% 2M NaCl in 20 mM NaH₂PO₄(pH 7.2). Check fractions. The p42-M.2 protein will elute in the 50%ethylene glycol fractions.

Example 3 Production and Purification of Baculovirus p42-K

The K1 type (p42-K) of the p42 antigen was constructed using theVietnam-Oak Knoll P. falciparum isolate (FVO). There are three parts tothis construct: a leader sequence, the p42-K coding region and thehistidine tag (FIG. 13). Restriction sites were incorporated into theprimers to enable a “sticky-end” ligation of the three fragments. Theleader sequence was altered from the original p42-M sequence such thatthree adenines were added three bases prior to the start site tooptimize the codon preference for baculovirus and insect cells as wellas the distance between the promoter sequence and the methionine startcodon (Ranjan et al. 1995. Virus Genes 9(2):149-153). Primers containingNarI and PstI restriction site sequences were used to amplify the 1,065base pair p42-K coding region corresponding to the Ala₁₃₄₉ to Ser₁₇₂₃(as numbered by Miller et al. 1993. Mol. Biochem. Parasitol 59(1):1-14.)of MSP-1 from genomic P. falciparum DNA. Primers containing BamHI andNarI restriction site sequences were used to amplify the 91 base pairleader sequence. Oligonucleotides containing PstI and KpnI restrictionsite sequences were made to generate the 25 base pair histidine tag. Allprimers and oligonucleotide sequences used for the p42-K constructs areshown in Table 1.

TABLE 1 PCR Primer Sequences used in the Construction of p42-K PrimerName Sequence (5′ to 3′) Leader F (SEQ ID NO: 10)ATTGGATCCACTAAAATGTGGAGCTGGAAG Leader R (SEQ ID NO: 11)TATGGCGCCCGCGGTGCAGAGTGTGGCTGT p42 F (SEQ ID NO: 12)TTAGGCGCCGCAGTAACTCCTTCCGTAATT p42 R (SEQ ID NO: 13)TAACTGCAGAAAATACCATCGAAAAGT His F (SEQ ID NO: 14)TAACTGCAGTCATCATCATCATCATCATTAATAAGGTACCGAG His R (SEQ ID NO: 15)ATAGGTACCTTATTAATGATGATGATGATGATGACTGCAGTTA Underlined sequencesrepresent restriction sites. Bold letters represent changes to theleader sequence.

The leader sequence of the BVp42 constructs and the p42-K coding regionfrom FVO malaria parasite DNA were amplified by a high fidelity PCRreaction containing both Taq and Pwo DNA polymerases. This allows forthe cloning of the leader and p42-K coding region into the transfervector without mutations, since Pwo DNA polymerase contain 3′ to 5′exonuclease proofreading activity. PCR reaction mixtures were brought upto 100 microliters with 200 micromolar of each dNTPs (Gibco BRL), 0.4micormolar primers, 10 microliters of DNA template, 0.75 units of highfidelity polymerase (Roche Molecular Biochemicals, Indianapolis, IN) and1X high fidelity reaction buffer (Roche Molecular Biochemicals). Inorder to amplify the leader sequence 1 cycle of 95° C. 5 min., and 35cycles of 95° C. 1 min., 56° C. 1min., and 72° C. 1 min were used. Forthe amplification of the p42-K coding region, 1 cycle of 95° C. 5 min,and 35 cycles of 95° C. 1 min., 56° C. 1 min., and 72° C. 2 min. wereused. The 1.1 kb p42-K PCR product was electrophoresed on a 1% SeaPlaque GTG (FMC) low melt agarose gel in 1X TAE and purified using theGeneclean III kit (Bio101, Vista, CA; ). The 90 base pair leader PCRproduct was electrophoresed on a 2% BioGel (Bio101 ) low melt agarosegel designed for small DNA fragments, excised, and purified using theMermaid Spin Kit (Bio 101, Inc., 1070 Jushua Way, Vista, CA 92083. Toconstruct the histidine tag, the His F and His R oligonucleotides werediluted to 5 μM in dH₂O. To anneal the oligonucleotides, equal volumeswere heated to 37° C. for 10 min. and at room temperature for 20 min.

The leader sequence, MSP-1 p42-K coding region and histidine tag wererestriction enzyme digested, purified by Geneclean III or Mermaid SpinKit as needed and ligated into pUC18 (FIG. 13). The ligation was used totransform competent bacteria prepared as described by Sambrook et al.(1989). Plasmids from colonies were isolated and analyzed. Once aplasmid with the insert was identified as being in the correctorientation, the insert was subcloned into the transfer vector pAC373and operably linked to the polyhedron promoter. Sequencing of the p42-Kconstruct revealed a minor discrepancy when compared to the publishedMSP-1 sequence (Miller et al. 1993. Mol Biochem Parasitol. 59(1):1-14.)A nucleic acid base mutation occurred at position 670 of the p42-Kcoding region, which resulted in an adenine to cytosine nucleotidesubstitution and in the CCA codon for proline instead of the ACA codonfor threonine. The complete nucleic acid and amino acid sequences aresown in FIG. 14.

Transfection of Spodoptera frugiperda (Sf9) cells with a pAC373 and theAutographa california nuclear polyhedrosis virus (AcMNPV) DNAsuccessfully produced recombinant virus. Supernatant containing therecombinant baculovirus was plaque assayed, plaque purified, andexpanded to obtain high tittered stocks. Plaque assay results show thatstocks contained titers to 1.6×10⁸ pfu/ml or recombinant baculovirus.These high titers stocks were used to infect HIGH FIVE™ insect cells ata multiplicity of infection (MOI) of about 5. Western blots (Sambrook etal. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press. Plainview, NY) showed that HIGH FIVE™ cells produce BVp42-K protein beginning at 18 hours post infection (data not shown). At42 hours the protein concentrations reached their peak levels of about3-4 mg/L and the insect supematants were harvested. Purification byaffinity chromatography using 5.2 Mabs or phenyl HIC followed by Ni-NTAremoved extraneous proteins. Analysis of purified recombinant protein bysilver stains and western blots showed that the recombinant proteincould be purified from insect cell supernatants and was present in highquantities (FIGS. 15 and 16).

Results of the BCA Protein Assay (Pierce, Rockford, IL.) showed thatreasonable amounts of purified protein are attainable. Affinitychromatography with monoclonal 5.2 antibodies yielded a proteinconcentration of 670 μg/ml at the peak aliquots. Phenyl HICchromatography followed by Ni-NTA chromatography yielded lowerconcentrations of protein (<100 μg/ml) at peak aliquots. The histidinetag does not seem to be binding efficiently to the Ni-NTA resin as canbe seen by the amount of residual material in western blots of thecolumn filtrate (FIG. 17). While the reason for this is unknown, it canbe speculated that the histidine tag is not fully exposed in BVp42-K.Interestingly, even the denaturation of the protein with 6 M urea didnot increase the recovery of purified protein.

Example 4 Comparison of Polyclonal Antibodies

Aotus lemurinus griseimembra monkeys (n=5) and New Zealand white rabbits(n=5) were immunized with BVp42 protein from the original BVp42construct (the MSP-1 p42 construct obtained from the FUP isolate of P.falciparum [representing the MAD 20 “M” allele]) as described by Changet al. 1996. Their antibodies were tested for reactivity withrecombinant BVp42 and BVp42-K using indirect ELISA. Absorbancies wereplotted on a scatter graph against the reciprocal of the antibodydilutions. Data points were adjusted into a linear relationship bychanging the reciprocal dilution axis into a logarithmic scale. Datapoints for which the reciprocal dilution was greater than 4×10⁶ were notused due to non-linearity at these dilutions. A regression line for thedata series was constructed and the equation of the line calculated. Theequation was solved for the X-intercept to determine the titer of serumsamples. A test for difference scores was performed on the data todetermine if any significant differences existed between antibody titersof BVp42 and BVp42-K (Koosis DJ. 1997. Statistics: A Self-TeachingGuide. John Wiley and Sons Inc., New York, N.Y.).

TABLE 2 ELISA Titer Comparison of Monkeys and Rabbits Immunized withBVp42 for BVp42 and BVp42-K BVp42 BVp42-K Ratio K/M Difference Monkey 3005 1310000 2340000 1.79 −1030000 82055 1060000 1660000 1.57 −60000081591 390000 360000 0.92 30000  3006 670000 480000 0.72 190000  414520000 620000 1.19 −100000 Average 790000 1092000 1.38 −302000 RabbitK192 2600000 2380000 0.92 220000 K191 1590000 1860000 1.17 −270000 K1711430000 1670000 1.17 −240000 K170 1210000 1380000 1.14 −170000 K1511730000 2100000 1.21 −370000 Average 1712000 1878000 1.10 −166000

The results showed that Aotus monkey sera reacted well with both thehomologous BVp42 and heterologous BVp42-K proteins (Table 2). Antibodytiters of BVp42 ranged from 3.9×10⁵ with monkey 81591 to 1.31×10⁶ withmonkey 3005. BVp42-K titers ranged from 3.6×10⁵ with monkey 81591 to2.34×10⁶ with monkey 3005. The average titer of the five monkeys withBVp42 was 7.9×10⁵ with the average titer with BVp42-K was 1.09×10⁶. The“t” test difference score result showed that antibody titers with BVp42were not significantly different from BVp42-K using a significance levelof 5% (a=0.05). Monkeys were able to recognize both MAD20 and K1 typesof BVp42 even though two-thirds of the protein differs in amino acidsequence. However, the K/M ratios between individual monkeys variedslightly indicating that there may be slight differences in thespecificity of antibody populations produced by individual animals.

Rabbit sera reacted equally well with the heterologous BVp42-K and thehomologous BVp42 (Table 2). Antibody titers of BVp42 ranged from1.21×10⁶ with rabbit K170 to 2.60×10⁶ with rabbit K192. BVp42-K titersranged from 1.38×10⁶ with rabbit K170 to 2.38×10⁶ with rabbit K192. Theaverage titer of the five rabbits with BVp42 was 1.71×10⁶ while theaverage titer with BVp42-K was 1.88×10⁶. Like the monkey results, “t”test difference scores show that antibody titers with BVp42 were notsignificantly different than BVp42-K using a significance level of 5%(α=0.05), emphasizing the fact the p42 dimorphism may not play animportant role in B cell antigen recognition and antibody specificity.The rabbit K/M ratios were not as varied as the monkey K/M ratios butwere similar averaging 1.1 and 1.4 respectively.

Example 5 Comparison of Monoclonal Antibodies

A panel of mouse Mabs made against the p42 region of MSP-1 was used toexamine potential conformational differences between BVp42 and BVp42-K.These antibodies have been previously characterized and the areas ofBVp42 reactivity established (Kaslow et al. 1993. Mol. Biochem.Parasitol. 63:283-2389). Mabs 4.2, 94-115, AD9.1, BC9.1, 91-33 and91-115 all recognize conformational epitopes found in the conserved 19kDa portion of BVp42. G13 is the only Mab found outside of the 19 KDaregion and recognizes a linear epitope. Results from western blots withthe various Mabs show that all of the antibodies react with both BVp42and BVp42-K except for G13 (FIGS. 18A and 18B). Western blots show thatG13 does not react with BVp42-K even though it has a high affinity forBVp42. Further tests with G13 (data not shown) using an ELISA, indicatethat G13 may react very slightly with BVp42-K but not near the extent itreacts with BVp42. This slight cross-reactivity was unexpected sinceG13's epitope is located in the variable region of the 33 kDa area ofBVp42 and was thought to be non-reactive with BVp42-K. Perhaps thedimorphism that exists in the N-terminal portion of the protein makesthis epitope only very slightly accessible to G13 or perhaps a similarbut not identical epitope exists on BVp42-K.

The positive results obtained with the other Mabs show that, despite itssequence differences at the N-terminus BVp42-K C-terminus seems to havethe same general structure as the 19 kDa region of MSP-1. The 19 kDaregion corresponding to block 17, which is considered to be a conservedregion of MSP-1, contains four amino acid sties that can vary betweenstrains. Antibodies used here (except for G13) recognize the conserved,conformational epitopes of the 19 kDa region (Kaslow et al. supra). Thisis important since the disulfide bridges found in the 19 kDa region playan important part in the tertiary structure of the protein. Moreimportantly, the polyclonal antibody data presented here and by Hui etal. 1993. Infect. Immun. 61:3403-3411) suggest that the vast majority ofBVp42 antibodies are made against the conserved portions of the protein,thus possibly down-playing the role of dimorphism in antibodyrecognition.

Example 6 Glycosylation Analysis

Visualization of BVp42-K with silver stains and western blots revealedthat two forms of the protein were being produced by insect cells.Contamination with BVp42 was easily ruled out because neither form ofBVp42-K reacted strongly with Mab G13, while BVp42 reacts strongly withthe antibody. Glycosylation tests revealed that both BVp42-M and BVp42-Kare glycosylated in the baculovirus expression system. Both silverstains and western blots reveal that digestion of the proteins withN-glycosidase F results in a shift of each of the proteins to a lowermolecular weight (FIG. 19A-B). More significantly, incubation ofN-glycosidase F with BVp42-K resulted in the resolution of the two formof the protein into one form of a smaller molecular weight. The shift toa lower molecular weight was less dramatic but still significant forBVp42. This difference corresponds to the presence of two potentialN-glycosylation sites in the BVp42-K p42 sequence and only one potentialN-glycosylation site in the BVp42 construct. Interestingly, thismolecular weight shift was not seen in parasite isolated MSP-1,confirming other reports that N-glycosylation may not occur in P.falciparum (Blackman et al. 1991. Mol. Biochem. Parasitol. 49:2934;Holder et al. 1992. Mem. Inst. Oswaldo Cruz 87:37-42).

Tunicamycin experiments also supported the presence of N-glycosylationfor both proteins in insect cells. Incubation of HIGH FIVE™ cells withtunicamycin during recombinant baculovirus infection with eitherconstruct resulted in the same molecular weight shift of the expressedprotein as seen with the N-glycosidase F (FIG. 20). Incubation ofBVp42-K with other glycosidases (O-glycosidase, β-galactosidase andneuraminidase) did not result in a molecular weight shift of the proteinvisualized by silver staining.

Example 7 Production and Purification of Yeast p42 (Yp42)

A yeast p42 construct was expressed in the alcohol dehydrogenase2/glyceraldehyde-3-phosphate dehydrogenase (ADH₂-GAPDH) regulatedexpression system of Saccharomyces cerevisiae and has been describedfully in Hui et al. 1991. J. Immunol. 147:3935-3941, expressingincorporated by reference. The p42 construct was based on the gp195coding region from Ala₁₃₃₃, to Ser₁₇₀₅ of the FUP isolate (Chang et al.1988. Exp. Parasitol. 67:1). The yeast p42 polypeptide (Yp42) waspurified using an affinity chromatography technique described bySiddiqui et al. 1987 for the purification of parasite gp195, except thatpolyclonal rabbit anti-gp195 IgG was used for antigen purificationinstead of anti-gp195 Mab 5.2, because the Yp42 protein could not berecovered in significant amounts using this Mab.

FIG. 2B shows silver stains (lane 1) and immunoblots (lane 2) of theaffinity chromatography purified Yp42. The silver stained antigens wereelectrophoresed in SDS polyacrylamide gels. The immunoblots were reactedwith a rabbit anti-parasite gp195 serum pool. In the silver stain, amajor species corresponding to Yp42 migrated as a 44 kDa molecule,although other minor bands were also present in varying amounts. In theimmunoblots, the purified Yp42 displayed major immunoreactive species atthe positions of the major protein bands, accompanied by minorreactivities with proteins of higher and lower molecular weight.

Example 8 Isolation of Purified, Parasite gp 195 with Selected Fragmentsthat Induce Substantially Complete Protection Against Plasmodiumfalciparum Challenge

A mixture of gp195 protein and certain of its processing fragments wereobtained from in vitro cultured parasites (P. falciparum Uganda PaloAlto strain) using monoclonal-antibody affinity chromatographyprocedures employing Mab 5.2 (Siddiqui et al. 1987); i.e. the mixture isenriched in the epitope for which Mab 5.2 is specific. Importantly, thismixture has been previously shown to be capable of inducingsubstantially complete protection against a homologous challenge ofPlasmodium falciparum in Aotus monkeys (Siddiqui et al. 1987).References in the examples below to “gp195” refer to the Mab5.2-purified mixture of gp195 enriched with certain of its processingfragments.

In summary, saponin-lysed parasites were extracted with 1% NP-40 and thelysate was clarified by ultracentrifugation. The extracts were passedthrough a Protein G Sepharose column covalently conjugated withgp195-specific Mab 5.2 (U.S. Pat. No. 4,897,354: expressly incorporatedby reference and deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md., USA 20852, on Jul. 17, 1986 underaccession number HB 9148). Mab 5.2 is specific for an antigenicdeterminant contained in p42, as demonstrated below.

After extensive washing to remove non-specifically bound material,specifically-bound proteins were eluted with 0.1 M glycine (pH 2.5) andneutralized with 1 M Tris-HCl (pH 8.0). The purity of the isolated gp195 was examined by SDS-PAGE followed by silver staining.

Example 9 In Vitro Inhibition Assay

Substantially complete parasite inhibition was found obtainable usingBVp42 as an immunogen in in vitro parasite growth inhibition assays.This level of inhibition has only been observed by us to be induced bythe Mab 5.2 affinity-purified mixture of gp195 and fragments (Hui et al.1987. Exp. Parasitol. 64:519). In vitro parasite growth inhibitionassays were performed by culturing parasites in the presence of immunerabbit serum using established methods (Hui et al. 1987. Exp. Parasitol.64:519). Briefly, parasites were cultured in the presence of 15%preimmune serum or immune rabbit serum obtained 14, 21, 28, and 35 daysafter the fourth immunization with BVp42 in Complete Freund's Adjuvant(indicated as 4D14, 4D21, 4D28 and 4D35 in Table 3). The startingparasitemia (S) in each of the experiments was 0.2%. Growth inhibitionwas calculated according to the following equation:

${{\%\mspace{20mu}{inhibition}} = {\frac{\left( {P - S} \right) - \left( {T - S} \right)}{\left( {P - S} \right)} \times 100\mspace{11mu}\%}},{where}$

-   P=% parasitemia of cultures containing 15% preimmune serum at 72    hours;-   T=% parasitemia of cultures containing 15% immune serum at 72 hours;    and-   S=starting % parasitemia of cultures at 0 (zero) hours.

Quaternary sera were used in inhibition assays. The correspondingpre-immune serum of each animal was used as a control. Parasite cultureswere synchronized by sorbitol lysis (Lambros et al. 1979. J. Parasitol.65:418) to select for late trophozoite and schizont stages. Infectederythrocytes were adjusted to an initial parasitemia of approximately0.2% and a hematocrit of 0.8% with fresh erythrocytes. Rabbit preimmuneor immune serum was added to a final concentration of 15%, and 200 μl ofthe culture suspension were added-in duplicate wells to a 96-wellmicrotiter plate. Cultures were incubated at 37° C. for 72 hours, andthe parasitemia was determined by microscopy. The experiment wasrepeated three times for each of the rabbits used (rabbits givenidentification nos. 131 and 132). Results are shown in Table 3.

Quaternary sera of rabbits immunized with Yp42 had no significant effecton in vitro parasite growth (data not shown). In contrast, significantinhibition was obtained with quaternary sera of several bleeding datesfrom both rabbits immunized with BVp42 (Table 3). Sera obtained fromlater bleedings of rabbit 132 (4D21, 4D28, 4D35) nearly completelyinhibited parasite growth; similar levels of inhibition have beenobserved previously only with antisera against Mab 5.2 purified,parasite gp195 (Hui et al. 1987).

TABLE 3 In Vitro Parasite Growth Inhibition Assay with Rabbit Anti-BVp42Sera % Parasitemia (% Inhibition) Rabbit Serum Expt. 1 Expt. 2 Expt. 3Anti-BVp42 (131): Preimmune 13.3     6.8   9.8   4D14 10.3 (23)  — —4D21 5.6 (58) 3.9 (44) 4.0 (60) 4D28 — 1.7 (77) 2.6 (75) 4D35 — 1.6 (79)1.9 (82) Anti-BVp42 (132): Preimmune 9.0 (11) 6.7   8.7   4D14 5.4 (47)— — 4D21 <0.1 (>99) 0.4 (98)  0.2 (100) 4D28 — 0.3 (98) 0.3 (99) 4D35 —0.3 (98) 0.9 (92)

Example 10 Determination of Immunogenicities and Cross-Reactivities ofRecombinant Polypeptides

A. ELISA Titers of Rabbits Immunized with Purified Parasite gp195Against gp195, BVp42 and Yeast p42.

Rabbits were immunized with BVp42, Yp42 or enriched gp195 mixture (i.e.enriched for gp195 and C-terminal containing processing fragmentsthrough Mab 5.2 affinity purification) to determine the immunogenicityof the recombinant polypeptides and the cross-reactivity ofanti-recombinant p42 antibodies with native gp195.

Rabbits given identification nos. 103, 104, 106 and 115 were immunizedwith purified, parasite gp195 emulsified in Complete Freund's Adjuvantas described (Hui et al. 1987). Rabbits 131 and 132 were immunizedintramuscularly four times at 21-day intervals with 50 μg of purifiedBVp42 in Complete Freund's Adjuvant. Rabbits 93 and 96 were immunizedintramuscularly five times at 21-day intervals with 50 μg of purifiedYp42 in Complete Freund's Adjuvant. The amount of mycobacteria wasreduced to one half of the first dose for the second immunization andone fourth of the first dose for subsequent immunizations, with thevolume being replaced with Incomplete Freund's Adjuvant. Rabbits werebled before immunization and weekly after each immunization.

Serum antibodies produced against enriched gp 195 mixture, BVp42, orYp42 were assayed by an enzyme-linked immunosorbent assay (Chang et al.1989; Chang et al. 1988) using the following technique. Vinyl plateswere coated with purified, parasite gp195 and fragments (0.08 μg/ml),recombinant BVp42 (0.08 μg/ml), or recombinant Yp42 (0.2 μg/ml) andwashed and blocked with 1% bovine serum albumin in borate bufferedsaline (BBS: 167 mM borate/134 mM NaCl, pH 8.0). Rabbit and mouse serawere serially diluted in 1% BSA/BBS, and human sera were seriallydiluted in phosphate-buffered saline (PBS: 150 mM sodium phosphate, 500mM NaCl, pH 7.4) containing 0.05% Tween 20, 1.5% powdered milk, 0.05%BSA and 0.05% thimerosal. Diluted sera were added to antigen-coatedwells and incubated for 1 hr at room temperature. Plates were washedwith BBS containing 0.5 M NaCl (HSBBS) (rabbit and mouse sera) or PBSwith 0.05% Tween 20 (human sera), and an appropriate dilution ofperoxidase-conjugated species-specific anti-IgG (heavy and light chainspecific) was added and incubated for 1 hr (anti-rabbit and anti-mouseIgG) or 2 hrs (anti-human IgG) at room temperature. Plates were washedin HSBBS and finally in BBS. One hundred microliters of peroxidasesubstrate solution [H₂O₂ and2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] were added to eachwell, and the absorbance value at 410 nm was determined with a Dynatech605 ELISA reader. The endpoint of ELISA titers for rabbit sera wasdesignated to be the serum dilution producing an absorbance value of0.2, which corresponded to twice the background O.D. reading. The ELISAendpoint titers for human sera was designated to be the serum dilutionproducing an absorbance value greater than 0.056, which was two standarddeviations above the average reading for normal human sera.

A comparison of the reactivity of the polyclonal antisera from rabbitsimmunized with the purified, enriched parasite gp195, BVp42, and Yp42 inan ELISA is shown in Table 4.

TABLE 4 ELISA Titers of Rabbits Immunized with Purified, Parasite gp195Against Parasite gp195, Baculovirus p42, and Yeast p42 Rabbit Serumgp195 Titer BVp42 Titer Yp42 Titer 103 1/140,000 1/5,400 1/1,000 1041/300,000 1/7,000 1/1,300 106 1/330,000 1/20,000 1/3,000 115 1/160,0001/4,200 1/800

The highest ELISA titers were obtained against parasite gp195, theimmunogen, with lower titers against BVp42, and the lowest titersagainst Yp42. These results are consistent with the expectation thatantibodies are induced by gp 195 outside of the p42 fragment, leading toa higher titer against the gp195 preparation than against BVp42.

B. ELISA and IFA Titers of Rabbits Immunized with BVp42 and Yeast p42Against Parasite gp195, BVp42 and Yp42.

Additional rabbits were immunized with purified BVp42 (nos. 131, 132) orpurified Yp42 (nos. 93, 96) to determine the immunogenicity of therecombinant polypeptides and the cross reactivity of anti-recombinantp42 antibodies with native gp195. Rabbit sera were tested by ELISA,parasite indirect immunofluorescence (IFA), and immunoblotting withparasite gp195.

The IFA procedure used was as follows. Assays were performed onacetone-fixed thin blood smears of schizonts and merozoites as described(Siddiqui et al. 1986. Infect. Immun. 52:314). Endpoint IFA titerscorresponded to the final serum dilution producing parasiteimmunofluorescence above background levels observed using preimmunerabbit sera.

Sera were obtained on the day indicated (day 14, 21, 28 or 35, indicatedas 4D14, etc. in Table 5 below) after four immunizations of rabbits with50 μg BVp42 or 50 μg Yp42. Serial dilutions of sera were titered forreactivity with plates coated with recombinant p42 (rp42) or parasitegp195. “rp42” corresponds to BVp42 for rabbit anti-BVp42 sera and Yp42for rabbit anti-Yp42 sera. The method of determining the ELISA endpointtiter for rabbit sera was as discussed above. Endpoint IFA titerscorrespond to the final serum dilution producing parasiteimmunofluorescence above preimmune serum backgrounds.

BVp42 proved to be highly immunogenic as shown in Table 5, inducingantibody titers comparable to those of rabbits immunized with purified,parasite gp195 (Table 2).

As shown in Table 5, ELISA titers were similar in assays utilizingplates coated with either purified, parasite gp195 or BVp42. Very highELISA titers were obtained late in the quaternary response (days 28 and35). Yp42 was less immunogenic than BVp42, inducing lower antibodytiters against the immunogen. In addition, the cross-reactivity ofanti-Yp42 antibodies with parasite gp195 in the ELISA was much lowerthan the cross-reactivity of anti-BVp42 antibodies. Typical merozoitesurface staining patterns were observed by IFA for the BVp42 antisera(data not shown), and IFA titers obtained after the fourth immunization(Table 5) reached levels exceeding those obtained by immunization withpurified, parasite gp 195 (data not shown). Yp42 induced much lower IFAtiters.

C. ELISA Inhibition Assay

In order to obtain an estimate of the degree of cross-reactivity ofanti-parasite gp 195 (enriched mixture) antibodies with BVp42 and Yp42,we performed an ELISA inhibition assay utilizing the various antigens asinhibitors. The ELISA inhibition assay measures the reduction inreactivity of anti-parasite gp195 sera with parasite gp195 antigen inthe presence of various concentrations of soluble parasite gp195 orrecombinant p42. The ELISA inhibition assay was performed by dilutingrabbit antisera to a point on the descending portion of the ELISAtitration curve. The diluted sera (rabbit anti-parasite gp195 sera 103,104, 106, and 115) were mixed with various concentrations of inhibitor(gp195, BVp42, or Yp42), incubated for 1 hour, and added to purified,parasite gp195-coated plates. Subsequent steps in the ELISA inhibitionassay were identical to the standard ELISA assay described in section Aabove.

TABLE 5 ELISA and IFA Titers of Rabbits Immunized with Baculovirus p42and Yeast p42 Against Parasite gp195, Baculovirus p42 and Yeast p42 rp42ELISA Gp195 ELISA Rabbit Serum Titer Titer IFA Titer Anti-BVp42 (131):4D21 1/100,000 1/30,000 1/25,600 4D28 1/120,000 1/130,000 1/51,200 4D351/98,000 1/160,000 1/204,800 Anti-BVp42 (132): 4D21 1/74,000 1/85,0001/51,200 4D28 1/120,000 1/300,000 1/102,400 4D35 1/88,000 1/300,0001/204,800 4D14 1/60,000 1/2,000 1/1,600 4D21 1/40,000 1/2,000 1/3,2004D28 1/50,000 1/3,000 1/6,400 Anti-Yp42 (96) 4D14 1/25,000 1/900 1/8004D21 1/23,000 1/800 1/3,200 4D28 1/30,000 1/700 1/6,400

The results are shown in FIG. 4. Soluble parasite gp195 completelyinhibited the binding of anti-gp 195 sera in this assay. A high level ofinhibition (82-92%) was also obtained with soluble BVp42. Much lowerlevels of inhibition were seen with soluble Yp42 (28-47%). The averageantigen concentration required to obtain 50% inhibition in The ELISA wassimilar for parasite gp195 (0.03 μg/ml) and BVp42 (0.04 μg/ml), while >5μg/ml Yp42 was required for 50% inhibition. These results suggest that amajority of antibodies against parasite gp195 also recognize BVp42, i.e.purified, parasite gp195 and BVp42 are highly cross-reactive.

D. Reactivity with Human Sera

The recognition of gp195 (without Mab 5.2 affinity purified fragments)and related recombinant polypeptides by serum antibodies of individualsfrom a malaria-endemic area of the Philippines have been analyzed byKramer and Oberst. Several individuals from this study were examined forantibody reactivity with parasite gp195 (Mab 5.2 affinity purified),BVp42 and Yp42. The results are shown in Table 6

TABLE 6 Reactivity of Human Sera from a Malaria-Endemic Area withPurified, Parasite gp195, the Baculovirus p42 Polypeptide, and the Yeastp42 Polypeptide gp195 ELISA BVp42 ELISA Yp42 ELISA Individual TiterTiter Titer 14270 1/100 1/200 neg. 14103 1/102,400 1/51,200 1/400 141221/102,400 1/51,200 1/400 14184 1/3,200 1/3,200 1/800 13563 1/200 1/1,600neg. 13691 1/51,200 1/12,800 1/200 14187 1/21,800 1/12,800 1/100

Serial serum dilutions were tested for reactivity with the plates coatedwith parasite gp195, BVp42 and Yp42. Endpoint titers were designated asthe serum dilution producing an O.D.>0.056, which was two standarddeviations above the average reading for normal human sera. There was anexcellent correlation between ELISA titers obtained with parasite gp195and BVp42 (Pearson's correlation coefficient r=0.96, p<0.001). A lowercorrelation was obtained for gp195 and Yp42 ELISA titers (Pearson'scorrelation coefficient r=0.86, p<0.01). Thus, BVp42 cross reacts withserum antibodies of humans from the malaria endemic area of thePhilippines.

E. Recognition of Conformational Determinants by Antibodies of RabbitsImmunized with Parasite gp195 and Recombinant p42 and by a gp195specific, Mab 5.2.

FIG. 3, Panels A and B show immunoblots of purified parasite gp195electrophoresed under nonreducing (panel A) or reducing (panel B)conditions in a 11.5% SDS-polyacrylamide gel. Immunoblots were reactedwith the following antibody preparations: lane 1, anti-gp195 Mab 5.2;lane 2, rabbit anti-parasite gp195; lane 3, rabbit anti-BVp42 (#131);lane 4, rabbit anti-BVp42 (#132); lane 5, rabbit anti-Yp42 (#93); lane6, rabbit anti-Yp42 (#96). Panel C: Immunoblots of BVp42 (lane 1,non-reduced; lane 2, reduced) and Yp42 (lane 3, non-reduced; lane 4,reduced) reacted with Mab 5.2.

The procedure for immunoblotting was as follows. Purified gp195 andBVp42 polypeptides were dissolved in Laemmli's buffer with or without2-mercaptoethanol as a reducing agent and separated on NaDodSO₄polyacrylamide gels (Laemmli. 1970. Nature 227:680). The separatedproteins were electrophoretically transferred to nitrocellulose (Towbinet al. 1979. Proc. Natl. Acad. Sci. USA 76:4350) and reacted with rabbitand mouse antisera as described by Chang et al., 1989.

FIGS. 3A-B, lane 1, show that Mab 5.2 is specific for a conformationaldeterminant of the p42 fragment of gp195 since reactivity with thisantibody was diminished by reduction of the parasite antigen. FIG. 3Blanes 2, 3, and 4 also demonstrates that reactivity with the p42processing fragments of both anti-gp 195 sera and anti-BVp42 sera wasmarkedly decreased when parasite gp 195 was electrophoresed underreducing conditions. This indicates that both these antisera primarilyrecognize disulfide, conformational determinants of parasite gp195. Incontrast, reactivity of anti-Yp42 sera, as shown in FIG. 3B (lanes 5 and6) was slightly enhanced for reduced gp195, and the reduced 19 kDaprocessing fragment was recognized by these sera. Thus, the anti-Yp42appears to recognize gp195 epitopes that are not disulfide-dependent.

The strong reactivity of BVp42 with Mab 5.2, as shown in FIG. 3C, lane1, and the loss of this reactivity by reduction of BVp42, as shown inFIG. 3C, lane 2, indicates that the conformation of BVp42 closelyresembles that of the native protein.

Example 11 Recognition of BVp42 by Antibodies from Congenic MouseStrains

It has previously been shown that responsiveness to purified, parasitegp195 is present in mice of diverse major histocompatibility complexmakeup (Chang et al. 1989). We determined whether the BVp42 antigenwould be similarly recognized by anti-gp195 antibodies from a panel ofcongenic mouse strains.

Sera of seven congenic mouse strains possessing different H-2 haplotypeson the B 10 background and that had been immunized with purified,parasite gp195 were tested for reactivity with the BVp42 antigen (FIG.5). Congenic mice of the following strain designations differing in H-2haplotype but sharing the C57BL/10 genetic background were immunized:C57BL/10 SnJ, B10.A/SgSnJ, B10.D2/nSnJ, B10.M/SN, B10.WB (69NS),B10.BR/SgSnJ, and B10.PL (Jackson Laboratories, Bar Harbor, Me.). Fivemice per group were immunized intraperitoneally four times at 2-weekintervals with 5 μg of purified, parasite gp195 emulsified in CompleteFreund's Adjuvant. Mice were bled before immunization, on days 7 and 10after the first immunization and on days 5, 7, and 14 after subsequentimmunizations.

All seven strains produced anti-gp195 antibodies recognizing epitopes ofBVp42 with some variation in titer among strains. Thus, similar toparasite gp195, individuals of diverse MHC haplotypes are capable ofproducing antibodies recognizing BVp42.

Example 12 Reactivity of Anti-BVp42 with Homologous vs. HeterologousAntigens

Since the gp42 processing fragment contains both conserved and allelicdeterminants, the reactivity of anti-BVp42 antibodies with homologousvs. heterologous gp195 antigens was characterized. Four parasiteisolates, FUP, FVO, Hond-1 and Pf857 were used and by Southern blotanalyses with allele specific oligonucleotide probes showed that FUP andPf857 belong to the MAD allele, and FVO and Hond-1 belong to the K1allele. Native gp195 antigens were purified by affinity chromatographyfrom FUP and FVO parasites. In ELISAs, identical titers and bindingcurves were obtained with anti-BVp42 antibodies using either FUP(homologous) or FVO (heterologous) gp195 as antigens. Similar resultswere obtained in indirect immunofluorescent assays with FVO and FUPmerozoites. More importantly, anti-BVp42 antibodies strongly orcompletely inhibited the in vitro parasite growth of the heterologousparasites (FVO and Hond-1) to the same degree as with the homologousparasites (FUP and Pf857).

Example 13

Immunogenicity of Various Adjuvant Formulation of BVp42 in Aotus monkeysAotus lemurinus griseimembra monkeys of karyotypes II and III with nohistory of previous P. falciparum exposure were screened for lack ofBVp42 antibody reactivity by enzyme-linked immunosorbent assay (ELISA),the ability of the sera to support in vitro growth of plasmodia, normalblood chemistry and hematology values, absence of blood and intestinalparasites, absence of cardiovascular abnormalities and overall goodhealth. Aotus monkeys passing this screening process were stratified bygender before being randomly assigned to the control or experimentalgroups. All animals were maintained at the University of HawaiiLaboratory Animal Service facility in accordance with NationalInstitutes of Health and institutional guidelines.

The recombinant BVp42 antigen is based on the C-terminal merozoitesurface protein-I (MSP 1) sequence of the Uganda-Palo Alto P. falciparumisolated (FUP) (Chang et al. 1988) which is closely related to the MAD20MSP1 allele. BVp42 corresponds to the p42 coding region from Ala₁₃₃₃ toSer₁₇₀₅ cloned into the Autographica californica nuclear polyhedrosisvirus polyhedron promoter-regulated expression system (Luckow et al.1988). A similar construct, designated BVp42-K, was generated consistingof the corresponding p42 coding region of the Vietnam-Oak Knoll isolatewhich is closely related in sequence to the Wellcome-K1 MSP1 allele (T.Nishimura, unpublished data). The recombinant p42 polypeptides wereisolated from culture supernates of baculovirus infected Trichoplusia nicells by immuno-affinity chromatography as previously described(Siddiqui et al. 1987). BVp42 purity was assessed by silver staining ofantigen preparations separated by polyacrylamide gel electrophoresis.

Native MSP-1 protein and its processing fragments were purified from invitro cultured parasites by immuno-affinity chromatography (Siddiqui etal. 1987).

Immunizations consisted of 100 μg of BVp42 in 0.25-0.5 ml ofbacteriostatic 0.9% saline solution (Abbott Laboratories, North Chicago,IL) formulated with the adjuvants: MF59 (Chiron Corporation), MTP-PE(CIBA-Geigy), STIMULON™ QS21 (Aquila Biophamaceuticals), or MONTANIDE™ISA51 oil-in water emulsion (Seppic Inc.) according to the manufacturersinstructions. In the case of MF59, immunizations were administered onweeks 0, 3, 6 and 9. An extended immunization protocol was used forMTP-PE+MF59 (MTP-PE: mutamyl tripeptide covalently linked to dipalmitoylphosphatidyl ethanolamine; MF-59: and oil-in-water emulsion containingsqualene, polysorbate 80 (Tween-20), sorbian trioleate (Span85), andthimersol) for which immunizations were administered on weeks 0, 8, 24and 33. Monkeys received 200 μg MTP-PE in 0.5 ml MF-59 for eachimmunization (Keitel et al. 1993. Vaccine 11:9090-913). For both QS-21and ISA51 formulations, immunizations were given on weeks 0, 4, 8, 17,and 21. Each dose was given intramuscularly into altemating left andright thigh areas. In the first experiment, approximately 3 ml bloodsamples were collected from the femoral vein 14 days after eachimmunization and, for experiment 2, blood samples were collected monthlybetween the secondary and tertiary immunizations. Unimmunized animals oranimal immunized with adjuvant alone served as controls.

TABLE 7 Experimental Groups Receiving various Adjuvant Formulation ofthe BVp42 Merozoite Surface Protein 1 Vaccine Adjuvant Group AdjuvantAnimal Nos. I MF-59 80140, 80315 II MTP-PE + MF-59 84017, 84033, 84034,84038 III QS21 3006, 414, 81591, 82055 IV Montanide ISA-51 3005, 81194,82115

All of the adjuvant formulations tested were generally well toleratedand produced no systemic side effects in vaccinated monkeys. Localeffects produced from immunization with BVp42/MF-59 included persistentinduration at the site of injection and enlarged inguinal lymph nodes.Animals immunized with BVp42/MTP-PE+MF59 also developed enlargedinguinal lymph nodes but no induration at the site of injection. Nolocal effects were observed at the site of injection or in the area ofthe regional lymph nodes for the BVp42/QS21 and BVp42/ISA51formulations.

FIGS. 8A-D present the kinetics of the antibody response to BVp42induced by immunization with the various adjuvant formulations.Significant boosting of antibody titer was observed after the secondimmunization with all four adjuvant formulations. Boosting was morevariable after subsequent immunizations. For BVp42 in MF59, the peaktiter for one animal (80315) was obtained after the third immunizationwhile the titer for another animal (80140) continued to increase afterthe fourth immunization. Similarly, animals immunized with BVp42 in theMTP-PE+MF59 formulation developed peak titers after the third or fourthimmunization although the titer of one animal (84017) reached itsmaximum after two immunizations, declining somewhat with subsequentimmunizations. All of the animals immunized with BVp42/QS21 orBVp42/ISA51 continued to develop increased antibody levels with repeatedimmunization and achieved peak antibody titers after four immunizations.

The cell mediated immune response induced by immunization was assessedby either antigen specific T-cell proliferation assays (FIG. 9) or bythe measurement of cytokine producing cells in antigen-stimulated andunstimulated T-cell cultures (FIGS. 10A-B). Significant antigen-specificT-cell proliferation was observed for peripheral blood mononuclear cellcultures of animals injected with the three formulations tested usingthis assay (FIG. 9: Groups I, II). Since cytokine production may be moreinformative than T-cell proliferation in assessing the type of T-cellresponse induced by the various adjuvant formulations, we also evaluatedintracellular cytokine production in subsequent vaccination studies.Peripheral blood of Aotus receiving adjuvant formulations II, III, andIV contained high number of interferon-gamma (IFNγ) producing cells(FIG. 10A). Significant numbers of IL-4 and IL-10 producing cells werealso detected in unstimulated peripheral blood CD4+ cells.

Effectiveness of T cell priming with the two adjuvant formulations,BVp42/ISA51 and BVp42/QS21, was evaluated by enumeration ofintracellular cytokine producing cells of these animals that wereunstimulated and BVp42 stimulated in vitro (Table 8). Prior toinitiation of immunization, the level of cytokine-producing cells in allanimals were <1% for IL-4 and IL-10 and 2% for IFNγ. Followingimmunization, significantly enhanced levels of cells producing the Th1cytokine IFNγ and the Th2 cytokines IL-4 and IL-10 were detected in allimmunized animals.

For animals immunized with BVp42/ISA51, a high percentage ofIFNγ-producing cells (11-21%) were detected in unstimulated lymphocytecultures of primed animals, indicating that immunization with thisformulation induced an increase in peripheral blood cells producing thisTh1 cytokine. Production of IL-10 was enhanced in BVp42-stimulatedcultures as compared to cultures incubated without antigen, indicatingthat Antigen-specific, primed T cells included a significant Th2,IL-10-producing component that could be expanded by BVp42 stimulation.In the other BVp42-stimulated cultures, the percentage of cytokineproducing cells either remained the same (IL-4) or was slightly reduced(IFNγ) as compared to unstimulated cultures containing no antigen.

In the case of Aotus immunized with BVp42/QS21, IFNγ was also thepredominant cytokine for most animals in terms of percentage of cytokineproducing cells in unstimulated cultures. However, both IL-4 and IL-10producing cells were enhanced upon antigen stimulation in vitro,consistent with effective priming of the Th2 antigen-specific populationwith this formulation.

TABLE 8 Cytokine Production in Aotus Vaccinated with BVp42 AdjuvantFormulations Unstimulated BVp42-stimulated Sample IFNγ IL4 IL10 IFNγ IL4IL10 BVp42/ISA51  3005 20.5^(a) 2.5 3 6 5.5 9 81194 11 1.5 1.5 4.5 1.55.5 82115 12 1.6 1.4 6.2 1.8 13.5 BVp42/QS21  414 2.5 1 2.5 2.3 2 5.1 3006 7 0.8 0.5 4.5 1.6 3 81591 5.5 0.6 0.4 3.5 1.5 2 82055 19.5 2.2 814.5 8 16.5 ^(a)numbers correspond to the % CD4+ cells

In summary, the cytokine data indicate that both BVp42 adjuvantformulations induced priming of Th1- and Th2-like lymphocyte populationsin vaccinated Aotus. While Th1-like IFNγ-producing cells comprised amajor population of circulating lymphocytes in these animals, inductionof antigen-specific Th2 cells producing IL-4 and IL-10 were alsoeffectively induced with these formulations.

Example 14 In Vitro Inhibition by Sera from Aotus Vaccinated with BVp42Adjuvant Formulations

Purified serum immunoglobulin of Aotus immunized with the variousadjuvant formulations were evaluated for biological activity in an invitro assay of P. falciparum growth inhibition (Chang et al., Inf.Immun. 64:253-261, 1996). The results of two separate experiments arepresented in Table 9. Preimmune immunoglobulin of all Aotus tested hadno effect on in vitro parasite growth. A high level of growth inhibition(94.3% and 92.3%) was observed for the animal displaying the highestlevel of protective immunity, Aotus 81194, which was immunized withBVp42 in ISA51. A lower level of inhibition (82115) was noted for asecond animal immunized with BVp42/ISA51 (Aotus 82115) although thisanimal showed no in vivo evidence of protective immunity. The thirdanimal immunized with the BVp42/ISA51 formulation (Aotus 3005) showed alow level of inhibitory activity in one of the two experiments. Aotusimmunized with the BVp42/QS21 formulation did not appear to producesignificant levels of inhibitory antibodies as only low levels ofinhibition were noted and inhibition was not consistent in duplicateexperiments. These results suggest that the in vitro growth inhibitionassay may be used as a marker to identify individuals with a high levelof immunity, as reflected by high levels of growth inhibition ofimmunoglobulin from Aotus 81194, and this may reflect one mechanism ofprotective immunity. However, this mechanism does not appear to beitself sufficient nor essential for protective immunity since (1) oneanimal displaying a detectable (albeit lower than 81194) level ofinhibition showed no evidence of protection against parasite infection,and (2) several animals showing partial protection against parasiteinfection (81591 and 3005) did not show significant or consistentinhibition of in vitro parasite growth.

TABLE 9 In vitro Inhibition by Sera from Aotus Vaccinated with BVp42Adjuvant Formulations Animal No. Expt #1* Expt. #2* (VaccineFormulation) Parasitemia (%) % Inh. Parasitemia (%) % Inh. Media Control3 3.8 417 (BVp42/CFA) 0.4 0.6 414 (BVp42/QS21) Preimmune 2.2 Neg. 2.8Neg. Immune 2.6 3.1 3005 (BVp42/ISA51) Preimmune 3 Neg. 3 21.4 Immune3.6 2.4 3006 (BVp42/QS21) Preimmune 2.8 Neg. 3.5 57.6 Immune 2.8 1.681194 (BVp42/ISA51) Preimmune 3.7 94.3 2.8 92.3 Immune 0.4 0.4 81591(BVp42/QS21) Preimmune 3.3 Neg. 3 Neg. Immune 3.2 3 82055 (BVp42/QS21)Preimmune 3.8 16.7 3.4 Neg. Immune 3.2 3.6 82115 (BVp42/ISA51) Preimmune3.3 29 3.8 41.7 Immune 2.4 2.3 Starting Parasitemia: 0.2%

Example 15 Aotus monkeys Immunized with BVp42 Adjuvant Formulations areProtected from a Lethal Challenge of Plasmodium falciparum

To generate the parasite challenge inoculum, a frozen stabilite of invivo-passaged FUP isolate P. falciparum was thawed and inoculated into amalaria-naive Aotus monkey (Expts. 1 and 3) or into an in vitro cultureof Aotus erythrocytes (Expt. 2). Peripheral blood containing parasitizederythrocytes was collected from infected animals or from overnightculture and adjusted to 7×10⁵ parasites per ml, and 0.5 ml was injectedinto the saphenous vein of control animals and experimental animals at 1week (Expt. 1), 3 weeks (Expt. 2) or 4 weeks (Expt. 3) after the finalimmunization. Parasitemias were monitored by Geimsa stained blood smearsof peripheral blood. The experimental endpoint for these vaccinationstudies was defined as either parasitemia >10% erythrocytes, anderythrocyte count of <3.0×10⁶/ml, or declining health as determined bythe attending veterinarian. Antimalarial therapy consisted of oralcholoroquine (10 mg/kg of body weight per day for 4 days) or mefloquine(25 mg/kg of body weight, at one time). The experiment was terminated byantimalarial drug treatment of remaining, untreated animals 6 weeksafter parasite challenge.

Prechallenge antibody titer values measured after the final immunizationfor the different adjuvant groups are presented in Table 10. High ELISAtiters (>500,000) were achieved in all adjuvant groups. The highestELISA titer (>10,000,000) was seen for an animal (81194) immunized withBVp42 in ISA51. At least one animal in each adjuvant group developed aprechallenge titer≧1,000,000. Consistently higher antibody titers werenoted for animals immunized with the MTP-PE+MF59 and ISA51 formulationsthan other adjuvant formulations. IFA titers followed a similar trend tothe ELISA titers, ranging from 1:1600 in animals with the lowest ELISAtiters to 1:6400 for the animal with the highest ELISA titer.

TABLE 10 Relationship of prechallenge anti-MSP1 antibody titers tocharacteristics of P. falciparum infection of Aotus immunized with BVp42in various adjuvant formulations. Prepatent Pretreatment ExperimentAnimal Prechallenge Antibody Titers Period Period No. No. ImmunogenELISA IFA (days) (days) 1 81570 — — — 3 8 80469 — — 4 12 80140 BVp42/1,600,000 1,600 3 10 80351 MF59 700,000 1,600 5 10 2 82016 — — — 3 1184028 7 13 84029 7 13 84017 BVp42/ 850,000 800 7 13 84033 MTP-PE +4,000,000 3,200 7 13 84034 MF59 3,000,000 3,200 7 13 84038 2,000,0003,200 7 13 3 416 — — — 5 7 419 5 7 81262 4 7 414 BVp42/ 700,000 3,200 714 3006 QS21 700,000 1,600 5 14 81591 550,000 3,200 4 —* 82055 1,000,0003,200 5 8 3005 BVp42/ 2,900,000 3,200 8 20 81194 ISA51 >10,000,000 6,40023 —* 82115 3,100,000 3,200 5 8 *No treatment administered duringexperiment; animals self-cured.

Experimental animals immunized with the various adjuvant formulationsalong with normal controls (expt. 1 & 3) or adjuvant controls (expt. 2)were administered a lethal, intravenous dose of Plasmodium falciparum(Uganda-Palo Alto isolate) after the final immunization. The course ofP. falciparum for these animals is presented in FIGS. 11A-D.

No significant differences in the course and final outcome of infectionwere seen between control and experimental Aotus immunized with theBVp42/MF59 (FIG. 11A) or the BVp42/MTP-PE+MF59 (FIG. 11C) formulations.The time intervals between parasite injection and detection ofperipheral blood parasitemia (prepatent period) and between challengeand drug-treatment (pretreatment period) were indistinguishable forcontrol and experimental animals receiving these two formulations (Table10).

In contrast, the course of P. falciparum infection was distinctlydifferent for Aotus immunized with both BVp42/QS21 and BVp42/ISA51formulations. For Aotus immunized with BVp42/QS21 (FIG. 11B), theprepatent period was similar to the normal controls but the course ofinfection and, in one instance, the outcome of infection were quitedifferent. While parasitemias of control animals rapidly rose totreatment levels (≧10%) within seven days after parasite challenge, thecourse of infection in three of the four animals in the experimentalgroup was much slower than the control group. These animals experienceda prolonged period of controlled parasite multiplication, resulting in asignificantly extended pretreatment period. For one animal in this group(81591), this phase of controlled parasitemia was followed by thegradual clearance of parasites from peripheral blood and self-cure ofthe infection.

The most striking results and strongest protection were obtained withAotus immunized with BVp42/ISA51 (FIG. 11D). The course of infection oftwo of the three animals in this group (3005 and 81194) was markedlydifferent from the controls. Aotus 3005 experienced a lengthenedprepatent period as compared to that of the control animals, after whichtime its parasitemia increased to moderate levels but was controlled foran extended period of time, similar to the QS21 group. While the trendof declining parasitemia suggested that this animal was in the processof clearing the parasite infection, it was drug-treated on day 20because of its impaired health status. The animal displaying the highestlevel of protective immunity was Aotus 81194 which had no detectableparasitemia until day 23 after challenge. Thereafter, its parasitemiaslowly increased, reaching a maximum of 1% on days 9-10 post-challenge.Following this peak in parasitemia, the parasite numbers abruptlydropped to low levels (<0.05%) for a week before disappearing fromperipheral circulation. Most notably, Aotus 81194 remained healthy andvigorous throughout the infection period, experiencing no reduction inits erythrocyte count (data not shown).

Table 11 presents the ELISA titers of animals immunized with BVp42/QS21and BVp42/ISA and challenged with P. falciparum with three differentsolid-phase antigens: recombinant FUP BVp42 (BVp42-M) representing thehomologous MAD20 MSP1 allele, recombinant FVO BVp42 (BVp42-K)representing the heterologous K1 MSP1 allele, and parasite-derived FUPMSP 1. In the case of Aotus immunized with BVp42/QS21, similar titerswere obtained using the homologous and heterologous BVp42 antigens andthere was a high level of cross reactivity of antibodies producedagainst the recombinant p42 MSP1 and the homologous MSP1 purified fromparasite extracts. The animal showing the highest level of immunitywithin this group (81591), as reflected by self-cure of the parasiteinfection, did not show a significant difference in specificity fromothers within the group. It is also interesting to note that the onlyanimal showing no evidence of protective immunity to parasite infectionin this group (82055), displayed a high ELISA titer for all threeantigens. Aotus 81194 immunized with BVp42/ISA51 developed the highesttiters against all three antigens and displayed the highest level ofprotective immunity. While ELISA titers were similar for another animalin the group displaying protective immunity (3005) and an unprotectedanimal (82115), there appeared to be a difference in specificity betweenthese two animals. Although the reactivity of Aotus 3005 was similar forthe homologous and heterologous BVp42 antigens (BVp42-M:BVp2-Kratio=1.3), the reactivity of Aotus 82115 was much lower for theheterologous than the homologous BVp42 antigen (BVp42-M:BVp42-Kratio=2.1). These results suggest that while the majority of antibodiesof Aotus 3005 (and Aotus 81194) may have been directed against conservedp42 epitopes, antibodies produced by Aotus 82115 may not be focused tothe same extent on these protective, conserved epitopes.

The results of the present study show for the first time that a MSP p42malaria antigen formulated with the adjuvants the STIMULON™ QS21 andMONTANIDE™ ISA51 adjuvants are capable of inducing a protective immuneresponse to P. falciparuminfection.

TABLE 11 Comparison of ELISA titers of Aotus immunized with BVp42/QS21and BVp42/ISA51 for homologous, recombinant BVp42-M, heterologous,recombinant BVp42-K, and homologous, parasite-purified MSP1. ELISA TiterAnimal No. Immunogen MSP1 BVp42^(a) BVp42-K^(b) M:K Ratio 414 BVp42/QS211,600,000 700,000 600,000 1.2 3006 700,000 700,000 460,000 1.5 81591900,000 550,000 360,000 1.5 82055 1,000,000 1,000,000 1,700,000 0.6 3005BVp42/ISA51 1,800,000 2,900,000 2,300,000 1.3 82115 1,700,000 3,100,0001,5000,000 2.1 81194 >10,000,000 >10,000,000 >10,000,000 ~1 ^(a)BVp42represents MSP-1 “M” allele ^(b)BVp42-K represents MSP-1 “K” allele

1. An isolated p42 nucleic acid encoding a p42 polypeptide from theC-terminal processing fragment of Plasmodium falciparum major merozoitesurface protein gp 195, wherein said p42 nucleic acid comprises anucleic acid which hybridizes under high stringency conditions to thecomplement of the nucleic acid of SEQ ID NO: 17; wherein said highstringency conditions comprise a salt concentration less than about 1.0M at about a pH of 7.0 to 8.3 and a temperature of at least about 30°C.; and wherein said encoded p42 polypeptide can induce ananti-plasmodium immune response in a primate.
 2. The isolated p42nucleic acid of claim 1, wherein said nucleic acid comprises thenucleotide sequence of SEQ ID NO:
 6. 3. An isolated p42 nucleic acidencoding a p42 polypeptide from the C-terminal processing fragment ofPlasmodium falciparum major merozoite surface protein gp 195, whereinsaid nucleic acid comprises the nucleotide sequence of SEQ ID NO:
 17. 4.An insect cell expression vector comprising the p42 nucleic acid ofclaim 1 or
 3. 5. The insect cell expression vector of claim 4, whereinsaid expression vector is a baculovirus vector.
 6. The insect cellexpression vector of claim 4, wherein said expression vector furthercomprises an optimized promoter operably linked to said nucleic acid. 7.The insect cell expression vector of claim 6, wherein said expressionvector is a baculovirus vector.
 8. An insect cell comprising the insectcell expression vector of claim
 4. 9. The insect cell of claim 8,wherein said insect cell is a Trichoplusia ni cell.
 10. An isolated p42nucleic acid encoding a p42 polypeptide from the C-terminal processingfragment of Plasmodium falciparum major merozoite surface protein gp195, wherein said nucleic acid comprises the nucleotides 1-1200 of thenucleotide sequence of SEQ ID NO: 17.